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Li X, Liu L, Lou H, Dong X, Hao S, Sun Z, Dou Z, Li H, Zhao W, Sun X, Liu X, Zhang Y, Yang B. Cardiomyocyte-specific long noncoding RNA Trdn-as induces mitochondrial calcium overload by promoting the m 6A modification of calsequestrin 2 in diabetic cardiomyopathy. Front Med 2025; 19:329-346. [PMID: 39821729 DOI: 10.1007/s11684-024-1102-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2024] [Accepted: 07/26/2024] [Indexed: 01/19/2025]
Abstract
Diabetic cardiomyopathy (DCM) is a medical condition characterized by cardiac remodeling and dysfunction in individuals with diabetes mellitus. Sarcoplasmic reticulum (SR) and mitochondrial Ca2+ overload in cardiomyocytes have been recognized as biological hallmarks in DCM; however, the specific factors underlying these abnormalities remain largely unknown. In this study, we aimed to investigate the role of a cardiac-specific long noncoding RNA, D830005E20Rik (Trdn-as), in DCM. Our results revealed the remarkably upregulation of Trdn-as in the hearts of the DCM mice and cardiomyocytes treated with high glucose (HG). Knocking down Trdn-as in cardiac tissues significantly improved cardiac dysfunction and remodeling in the DCM mice. Conversely, Trdn-as overexpression resulted in cardiac damage resembling that observed in the DCM mice. At the cellular level, Trdn-as induced Ca2+ overload in the SR and mitochondria, leading to mitochondrial dysfunction. RNA-seq and bioinformatics analyses identified calsequestrin 2 (Casq2), a primary calcium-binding protein in the junctional SR, as a potential target of Trdn-as. Further investigations revealed that Trdn-as facilitated the recruitment of METTL14 to the Casq2 mRNA, thereby enhancing the m6A modification of Casq2. This modification increased the stability of Casq2 mRNA and subsequently led to increased protein expression. When Casq2 was knocked down, the promoting effects of Trdn-as on Ca2+ overload and mitochondrial damage were mitigated. These findings provide valuable insights into the pathogenesis of DCM and suggest Trdn-as as a potential therapeutic target for this condition.
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Affiliation(s)
- Xiaohan Li
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Ling Liu
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Han Lou
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Xinxin Dong
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Shengxin Hao
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Zeqi Sun
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Zijia Dou
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Huimin Li
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Wenjie Zhao
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Xiuxiu Sun
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Xin Liu
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China
| | - Yong Zhang
- State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150081, China.
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China.
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China.
| | - Baofeng Yang
- Department of Pharmacology (SKLFZCD, State Key Laboratory-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin, 150081, China.
- Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin, 150081, China.
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Magyar ZÉ, Bauer J, Bauerová-Hlinková V, Jóna I, Gaburjakova J, Gaburjakova M, Almássy J. Eu 3+ detects two functionally distinct luminal Ca 2+ binding sites in ryanodine receptors. Biophys J 2023; 122:3516-3531. [PMID: 37533257 PMCID: PMC10502479 DOI: 10.1016/j.bpj.2023.07.029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2023] [Revised: 06/26/2023] [Accepted: 07/31/2023] [Indexed: 08/04/2023] Open
Abstract
Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane's electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate.
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Affiliation(s)
- Zsuzsanna É Magyar
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Jacob Bauer
- Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
| | | | - István Jóna
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Jana Gaburjakova
- Institute of Molecular Physiology and Genetics, Centre of Biosciences, Slovak Academy of Sciences, Bratislava, Slovakia
| | - Marta Gaburjakova
- Institute of Molecular Physiology and Genetics, Centre of Biosciences, Slovak Academy of Sciences, Bratislava, Slovakia
| | - János Almássy
- Department of Physiology, Semmelweis University, Budapest, Hungary.
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Murzilli S, Serano M, Pietrangelo L, Protasi F, Paolini C. Structural Adaptation of the Excitation-Contraction Coupling Apparatus in Calsequestrin1-Null Mice during Postnatal Development. BIOLOGY 2023; 12:1064. [PMID: 37626950 PMCID: PMC10452101 DOI: 10.3390/biology12081064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Revised: 07/21/2023] [Accepted: 07/26/2023] [Indexed: 08/27/2023]
Abstract
The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.
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Affiliation(s)
- Stefania Murzilli
- Department of Neuroscience, Imaging and Clinical Sciences (DNISC), Center for Advanced Studies and Technology (CAST), University G. d’Annunzio of Chieti-Pescara, 66100 Chieti, Italy;
| | - Matteo Serano
- Department of Medicine and Aging Sciences (DMSI), Center for Advanced Studies and Technology (CAST), University G. d’Annunzio of Chieti-Pescara, 66100 Chieti, Italy; (M.S.); (L.P.); (F.P.)
| | - Laura Pietrangelo
- Department of Medicine and Aging Sciences (DMSI), Center for Advanced Studies and Technology (CAST), University G. d’Annunzio of Chieti-Pescara, 66100 Chieti, Italy; (M.S.); (L.P.); (F.P.)
| | - Feliciano Protasi
- Department of Medicine and Aging Sciences (DMSI), Center for Advanced Studies and Technology (CAST), University G. d’Annunzio of Chieti-Pescara, 66100 Chieti, Italy; (M.S.); (L.P.); (F.P.)
| | - Cecilia Paolini
- Department of Neuroscience, Imaging and Clinical Sciences (DNISC), Center for Advanced Studies and Technology (CAST), University G. d’Annunzio of Chieti-Pescara, 66100 Chieti, Italy;
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Protasi F, Girolami B, Serano M, Pietrangelo L, Paolini C. Ablation of Calsequestrin-1, Ca 2+ unbalance, and susceptibility to heat stroke. Front Physiol 2022; 13:1033300. [PMID: 36311237 PMCID: PMC9598425 DOI: 10.3389/fphys.2022.1033300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 09/20/2022] [Indexed: 12/05/2022] Open
Abstract
Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.
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Affiliation(s)
- Feliciano Protasi
- Center for Advanced Studies and Technology, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
- Department of Medicine and Aging Sciences, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
| | - Barbara Girolami
- Center for Advanced Studies and Technology, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
- Department of Medicine and Aging Sciences, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
| | - Matteo Serano
- Center for Advanced Studies and Technology, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
- Department of Medicine and Aging Sciences, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
| | - Laura Pietrangelo
- Center for Advanced Studies and Technology, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
- Department of Medicine and Aging Sciences, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
| | - Cecilia Paolini
- Center for Advanced Studies and Technology, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
- Department of Neuroscience, Imaging and Clinical Sciences, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
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Neumann J, Bödicker K, Buchwalow IB, Schmidbaur C, Ramos G, Frantz S, Hofmann U, Gergs U. Effects of acute ischemia and hypoxia in young and adult calsequestrin (CSQ2) knock-out and wild-type mice. Mol Cell Biochem 2022; 477:1789-1801. [PMID: 35312907 PMCID: PMC9068673 DOI: 10.1007/s11010-022-04407-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2021] [Accepted: 03/03/2022] [Indexed: 11/26/2022]
Abstract
Calsequestrin (CSQ2) is the main Ca2+-binding protein in the sarcoplasmic reticulum of the mammalian heart. In order to understand the function of calsequestrin better, we compared two age groups (young: 4-5 months of age versus adult: 18 months of age) of CSQ2 knock-out mice (CSQ2(-/-)) and littermate wild-type mice (CSQ2(+/+)). Using echocardiography, in adult mice, the basal left ventricular ejection fraction and the spontaneous beating rate were lower in CSQ2(-/-) compared to CSQ2(+/+). The increase in ejection fraction by β-adrenergic stimulation (intraperitoneal injection of isoproterenol) was lower in adult CSQ2(-/-) versus adult CSQ2(+/+). After hypoxia in vitro (isolated atrial preparations) by gassing the organ bath buffer with 95% N2, force of contraction in electrically driven left atria increased to lower values in young CSQ2(-/-) than in young CSQ2(+/+). In addition, after global ischemia and reperfusion (buffer-perfused hearts according to Langendorff; 20-min ischemia and 15-min reperfusion), the rate of tension development was higher in young CSQ2(-/-) compared to young CSQ2(+/+). Finally, we evaluated signs of inflammation (immune cells, autoantibodies, and fibrosis). However, whereas no immunological alterations were found between all investigated groups, pronounced fibrosis was found in the ventricles of adult CSQ2(-/-) compared to all other groups. We suggest that in young mice, CSQ2 is important for cardiac performance especially in isolated cardiac preparations under conditions of impaired oxygen supply, but with differences between atrium and ventricle. Lack of CSQ2 leads age dependently to fibrosis and depressed cardiac performance in echocardiographic studies.
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Affiliation(s)
- Joachim Neumann
- Institute for Pharmacology and Toxicology, Medical Faculty, Martin Luther University Halle-Wittenberg, 06097 Halle, Germany
- Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Medizinische Fakultät, Magdeburger Str. 4, 06112 Halle, Germany
| | - Konrad Bödicker
- Institute for Pharmacology and Toxicology, Medical Faculty, Martin Luther University Halle-Wittenberg, 06097 Halle, Germany
| | | | - Constanze Schmidbaur
- Institute for Pharmacology and Toxicology, Medical Faculty, Martin Luther University Halle-Wittenberg, 06097 Halle, Germany
| | - Gustavo Ramos
- Department of Internal Medicine and Comprehensive Heart Failure Center, University Hospital Würzburg, 97080 Würzburg, Germany
| | - Stefan Frantz
- Department of Internal Medicine and Comprehensive Heart Failure Center, University Hospital Würzburg, 97080 Würzburg, Germany
| | - Ulrich Hofmann
- Department of Internal Medicine and Comprehensive Heart Failure Center, University Hospital Würzburg, 97080 Würzburg, Germany
| | - Ulrich Gergs
- Institute for Pharmacology and Toxicology, Medical Faculty, Martin Luther University Halle-Wittenberg, 06097 Halle, Germany
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Quantification of the calcium signaling deficit in muscles devoid of triadin. PLoS One 2022; 17:e0264146. [PMID: 35213584 PMCID: PMC8880904 DOI: 10.1371/journal.pone.0264146] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Accepted: 02/03/2022] [Indexed: 11/24/2022] Open
Abstract
Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The “hump” phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.
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Shi Y, Fan W, Xu M, Lin X, Zhao W, Yang Z. Critical role of Znhit1 for post-natal heart function and vacuolar cardiomyopathy. JCI Insight 2022; 7:148752. [PMID: 35167494 PMCID: PMC8986070 DOI: 10.1172/jci.insight.148752] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Accepted: 02/10/2022] [Indexed: 11/21/2022] Open
Abstract
Ca2+ is critical for cardiac electrical conduction and contractility, and aberrant Ca2+ homeostasis causes arrhythmia and heart failure. Chromatin remodeling modulates gene expression involved in cardiac sarcomere assembly and postnatal heart function. However, the chromatin-remodeling regulatory mechanism of cardiac Ca2+ homeostasis is unknown. Here, we found that Znhit1, a core subunit of the SRCAP remodeling complex, was essential for heart function. Deletion of Znhit1 in postnatal hearts of mice resulted in arrhythmia, idiopathic vacuolar cardiomyopathy, rapid heart failure, and premature sudden death. In addition, the level of Casq1, a sarcoplasmic reticulum Ca2+ regulatory protein, was massively elevated while SERCA2a showed reduced protein level. Mechanistically, the Znhit1 modulated the expression of Casq1 and SERCA2a by depositing H2A.Z at their promoters. Deletion of Casq1 could substantially alleviate the vacuolar formation in Znhit1Casq1 KO mice. These findings demonstrate that Znhit1 is required for postnatal heart function and maintains cardiac Ca2+ homeostasis and that accumulation of Casq1 might be a causative factor for vacuolar cardiomyopathy.
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Affiliation(s)
- Yingchao Shi
- Model Animal Research Center, Nanjing University, Nanjing, China
| | - Wenli Fan
- Model Animal Research Center, Nanjing University, Nanjing, China
| | - Mingjie Xu
- Model Animal Research Center, Nanjing University, Nanjing, China
| | - Xinhua Lin
- State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
| | - Wukui Zhao
- Model Animal Research Center, Nanjing University, Nanjing, China
| | - Zhongzhou Yang
- Model Animal Research Center, Nanjing University, Nanjing, China
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Nusier M, Shah AK, Dhalla NS. Structure-Function Relationships and Modifications of Cardiac Sarcoplasmic Reticulum Ca2+-Transport. Physiol Res 2022; 70:S443-S470. [DOI: 10.33549/physiolres.934805] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Sarcoplasmic reticulum (SR) is a specialized tubular network, which not only maintains the intracellular concentration of Ca2+ at a low level but is also known to release and accumulate Ca2+ for the occurrence of cardiac contraction and relaxation, respectively. This subcellular organelle is composed of several phospholipids and different Ca2+-cycling, Ca2+-binding and regulatory proteins, which work in a coordinated manner to determine its function in cardiomyocytes. Some of the major proteins in the cardiac SR membrane include Ca2+-pump ATPase (SERCA2), Ca2+-release protein (ryanodine receptor), calsequestrin (Ca2+-binding protein) and phospholamban (regulatory protein). The phosphorylation of SR Ca2+-cycling proteins by protein kinase A or Ca2+-calmodulin kinase (directly or indirectly) has been demonstrated to augment SR Ca2+-release and Ca2+-uptake activities and promote cardiac contraction and relaxation functions. The activation of phospholipases and proteases as well as changes in different gene expressions under different pathological conditions have been shown to alter the SR composition and produce Ca2+-handling abnormalities in cardiomyocytes for the development of cardiac dysfunction. The post-translational modifications of SR Ca2+ cycling proteins by processes such as oxidation, nitrosylation, glycosylation, lipidation, acetylation, sumoylation, and O GlcNacylation have also been reported to affect the SR Ca2+ release and uptake activities as well as cardiac contractile activity. The SR function in the heart is also influenced in association with changes in cardiac performance by several hormones including thyroid hormones and adiponectin as well as by exercise-training. On the basis of such observations, it is suggested that both Ca2+-cycling and regulatory proteins in the SR membranes are intimately involved in determining the status of cardiac function and are thus excellent targets for drug development for the treatment of heart disease.
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Affiliation(s)
| | | | - NS Dhalla
- Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen, Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6 Canada.
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The function and regulation of calsequestrin-2: implications in calcium-mediated arrhythmias. Biophys Rev 2022; 14:329-352. [PMID: 35340602 PMCID: PMC8921388 DOI: 10.1007/s12551-021-00914-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 11/14/2021] [Indexed: 01/09/2023] Open
Abstract
Cardiac arrhythmias are life-threatening events in which the heart develops an irregular rhythm. Mishandling of Ca2+ within the myocytes of the heart has been widely demonstrated to be an underlying mechanism of arrhythmogenesis. This includes altered function of the ryanodine receptor (RyR2)-the primary Ca2+ release channel located to the sarcoplasmic reticulum (SR). The spontaneous leak of SR Ca2+ via RyR2 is a well-established contributor in the development of arrhythmic contractions. This leak is associated with increased channel activity in response to changes in SR Ca2+ load. RyR2 activity can be regulated through several avenues, including interactions with numerous accessory proteins. One such protein is calsequestrin-2 (CSQ2), which is the primary Ca2+-buffering protein within the SR. The capacity of CSQ2 to buffer Ca2+ is tightly associated with the ability of the protein to polymerise in response to changing Ca2+ levels. CSQ2 can itself be regulated through phosphorylation and glycosylation modifications, which impact protein polymerisation and trafficking. Changes in CSQ2 modifications are implicated in cardiac pathologies, while mutations in CSQ2 have been identified in arrhythmic patients. Here, we review the role of CSQ2 in arrhythmogenesis including evidence for the indirect and direct regulation of RyR2 by CSQ2, and the consequences of a loss of functional CSQ2 in Ca2+ homeostasis and Ca2+-mediated arrhythmias. Supplementary Information The online version contains supplementary material available at 10.1007/s12551-021-00914-6.
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Hayter EA, Wehrens SMT, Van Dongen HPA, Stangherlin A, Gaddameedhi S, Crooks E, Barron NJ, Venetucci LA, O'Neill JS, Brown TM, Skene DJ, Trafford AW, Bechtold DA. Distinct circadian mechanisms govern cardiac rhythms and susceptibility to arrhythmia. Nat Commun 2021; 12:2472. [PMID: 33931651 PMCID: PMC8087694 DOI: 10.1038/s41467-021-22788-8] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 03/26/2021] [Indexed: 02/02/2023] Open
Abstract
Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.
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Affiliation(s)
- Edward A Hayter
- Centre for Biological Timing, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | - Sophie M T Wehrens
- Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK
| | - Hans P A Van Dongen
- Sleep and Performance Research Center, Washington State University, Spokane, WA, USA
- Elson S. Floyd College of Medicine, Washington State University, Spokane, WA, USA
| | | | - Shobhan Gaddameedhi
- Department of Biological Sciences, Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, USA
| | - Elena Crooks
- Sleep and Performance Research Center, Washington State University, Spokane, WA, USA
- Department of Physical Therapy, Eastern Washington University, Spokane, WA, USA
| | - Nichola J Barron
- Centre for Biological Timing, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | - Luigi A Venetucci
- Unit of Clinical Physiology, Manchester Academic Health Science Centre, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | | | - Timothy M Brown
- Centre for Biological Timing, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | - Debra J Skene
- Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK
| | - Andrew W Trafford
- Centre for Biological Timing, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
- Unit of Clinical Physiology, Manchester Academic Health Science Centre, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | - David A Bechtold
- Centre for Biological Timing, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
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11
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Yang M, Yan J, Wu A, Zhao W, Qin J, Pogwizd SM, Wu X, Yuan S, Ai X. Alterations of housekeeping proteins in human aged and diseased hearts. Pflugers Arch 2021; 473:351-362. [PMID: 33638007 PMCID: PMC10468297 DOI: 10.1007/s00424-021-02538-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2020] [Revised: 01/20/2021] [Accepted: 02/04/2021] [Indexed: 01/10/2023]
Abstract
Pathological remodeling includes alterations of ion channel function and calcium homeostasis and ultimately cardiac maladaptive function during the process of disease development. Biochemical assays are important approaches for assessing protein abundance and post-translational modification of ion channels. Several housekeeping proteins are commonly used as internal controls to minimize loading variabilities in immunoblotting protein assays. Yet, emerging evidence suggests that some housekeeping proteins may be abnormally altered under certain pathological conditions. However, alterations of housekeeping proteins in aged and diseased human hearts remain unclear. In the current study, immunoblotting was applied to measure three commonly used housekeeping proteins (β-actin, calsequestrin, and GAPDH) in well-procured human right atria (RA) and left ventricles (LV) from diabetic, heart failure, and aged human organ donors. Linear regression analysis suggested that the amounts of linearly loaded total proteins and quantified intensity of total proteins from either Ponceau S (PS) blot-stained or Coomassie Blue (CB) gel-stained images were highly correlated. Thus, all immunoblotting data were normalized with quantitative CB or PS data to calibrate potential loading variabilities. In the human heart, β-actin was reduced in diabetic RA and LV, while GAPDH was altered in aged and diabetic RA but not LV. Calsequestrin, an important Ca2+ regulatory protein, was significantly changed in aged, diabetic, and ischemic failing hearts. Intriguingly, expression levels of all three proteins were unchanged in non-ischemic failing human LV. Overall, alterations of human housekeeping proteins are heart chamber specific and disease context dependent. The choice of immunoblotting loading controls should be carefully evaluated. Usage of CB or PS total protein analysis could be a viable alternative approach for some complicated pathological specimens.
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Affiliation(s)
- Mei Yang
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Jiajie Yan
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Aimee Wu
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Weiwei Zhao
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Jin Qin
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Steven M Pogwizd
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294, USA
| | - Xin Wu
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA
| | - Shengtao Yuan
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, 210009, China.
| | - Xun Ai
- Department of Physiology and Biophysics, Rush University Medical Center, 1750 West Harrison St. 1255 Jelke South, Chicago, IL, 60612, USA.
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12
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Zhang XH, Morad M. Ca 2+ signaling of human pluripotent stem cells-derived cardiomyocytes as compared to adult mammalian cardiomyocytes. Cell Calcium 2020; 90:102244. [PMID: 32585508 PMCID: PMC7483365 DOI: 10.1016/j.ceca.2020.102244] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Revised: 06/10/2020] [Accepted: 06/11/2020] [Indexed: 12/23/2022]
Abstract
Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) have been extensively used for in vitro modeling of human cardiovascular disease, drug screening and pharmacotherapy, but little rigorous studies have been reported on their biophysical or Ca2+ signaling properties. There is also considerable concern as to the level of their maturity and whether they can serve as reliable models for adult human cardiac myocytes. Ultrastructural difference such as lack of t-tubular network, their polygonal shapes, disorganized sarcomeric myofilament, and their rhythmic automaticity, among others, have been cited as evidence for immaturity of hiPSC-CMs. In this review, we will deal with Ca2+ signaling, its regulation, and its stage of maturity as compared to the mammalian adult cardiomyocytes. We shall summarize the data on functional aspects of Ca2+signaling and its parameters that include: L-type calcium channel (Cav1.2), ICa-induced Ca2+release, CICR, and its parameters, cardiac Na/Ca exchanger (NCX1), the ryanodine receptors (RyR2), sarco-reticular Ca2+pump, SERCA2a/PLB, and the contribution of mitochondrial Ca2+ to hiPSC-CMs excitation-contraction (EC)-coupling as compared with adult mammalian cardiomyocytes. The comparative studies suggest that qualitatively hiPSC-CMs have similar Ca2+signaling properties as those of adult cardiomyocytes, but quantitative differences do exist. This review, we hope, will allow the readers to judge for themselves to what extent Ca2+signaling of hiPSC-CMs represents the adult form of this signaling pathway, and whether these cells can be used as good models of human cardiomyocytes.
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Affiliation(s)
- Xiao-Hua Zhang
- Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, Clemson University, Charleston SC, United States
| | - Martin Morad
- Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, Clemson University, Charleston SC, United States.
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13
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Rossi D, Gamberucci A, Pierantozzi E, Amato C, Migliore L, Sorrentino V. Calsequestrin, a key protein in striated muscle health and disease. J Muscle Res Cell Motil 2020; 42:267-279. [PMID: 32488451 DOI: 10.1007/s10974-020-09583-6] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Revised: 05/22/2020] [Accepted: 05/26/2020] [Indexed: 10/24/2022]
Abstract
Calsequestrin (CASQ) is the most abundant Ca2+ binding protein localized in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. The genome of vertebrates contains two genes, CASQ1 and CASQ2. CASQ1 and CASQ2 have a high level of homology, but show specific patterns of expression. Fast-twitch skeletal muscle fibers express only CASQ1, both CASQ1 and CASQ2 are present in slow-twitch skeletal muscle fibers, while CASQ2 is the only protein present in cardiomyocytes. Depending on the intraluminal SR Ca2+ levels, CASQ monomers assemble to form large polymers, which increase their Ca2+ binding ability. CASQ interacts with triadin and junctin, two additional SR proteins which contribute to localize CASQ to the junctional region of the SR (j-SR) and also modulate CASQ ability to polymerize into large macromolecular complexes. In addition to its ability to bind Ca2+ in the SR, CASQ appears also to be able to contribute to regulation of Ca2+ homeostasis in muscle cells. Both CASQ1 and CASQ2 are able to either activate and inhibit the ryanodine receptors (RyRs) calcium release channels, likely through their interactions with junctin and triadin. Additional evidence indicates that CASQ1 contributes to regulate the mechanism of store operated calcium entry in skeletal muscle via a direct interaction with the Stromal Interaction Molecule 1 (STIM1). Mutations in CASQ2 and CASQ1 have been identified, respectively, in patients with catecholamine-induced polymorphic ventricular tachycardia and in patients with some forms of myopathy. This review will highlight recent developments in understanding CASQ1 and CASQ2 in health and diseases.
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Affiliation(s)
- Daniela Rossi
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy.
| | - Alessandra Gamberucci
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Enrico Pierantozzi
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Caterina Amato
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Loredana Migliore
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Vincenzo Sorrentino
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
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14
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Gozalo AS, Zerfas PM, Elkins WR, Gieseck RL. Retrospective Study of Intercalated Disk Defects Associated with Dilated Cardiomyopathy, Atrial Thrombosis, and Heart Failure in BALB/c Mice Deficient in IL4 Receptor α. Comp Med 2020; 70:266-276. [PMID: 32384942 PMCID: PMC7287387 DOI: 10.30802/aalas-cm-19-000059] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Revised: 07/01/2019] [Accepted: 10/22/2019] [Indexed: 12/21/2022]
Abstract
An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of various ages, and extensive testing (microbiology, serology, histopathology) failed to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or, potentially, the Cre-loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.
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Affiliation(s)
- Alfonso S Gozalo
- Comparative Medicine Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland;,
| | - Patricia M Zerfas
- Pathology Service, Office of Research Services, National Institutes of Health, Bethesda, Maryland
| | - William R Elkins
- Comparative Medicine Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - Richard L Gieseck
- Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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15
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Hara T, Yamamura T, Murakami-Asahina M, Matsumoto H, Takeyama M, Kanagawa R, Nishimoto T. Development of a novel murine heart failure model overexpressing human renin and angiotensinogen. FEBS Open Bio 2020; 10:718-725. [PMID: 32056390 PMCID: PMC7193172 DOI: 10.1002/2211-5463.12810] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Revised: 01/14/2020] [Accepted: 02/12/2020] [Indexed: 01/18/2023] Open
Abstract
Renin is the rate‐limiting enzyme of the renin–angiotensin system cascade, which drives the pathophysiological progression of heart failure. Species differences in the amino acid sequence of the catalytic domain of renin limit evaluations of the potency and efficacy of human renin inhibitors in animal models, and a high dose of inhibitors is usually needed to show its organ‐protective effects in rodents. In the present study, we developed a novel murine heart failure model (triple‐tg) to enable us to evaluate the cardioprotective effect of renin inhibitors at more relevant doses for humans, by cross‐breeding calsequestrin transgenic (CSQ‐tg) mice with human renin and human angiotensinogen double‐transgenic mice. The triple‐tg mice exhibited increased plasma renin activity, worsened cardiac hypertrophy, and higher mortality compared to CSQ‐tg mice. Triple‐tg mice treated with 10 mg·kg−1 of TAK‐272 (imarikiren/SCO‐272), an orally active direct renin inhibitor, exhibited improvements in heart failure phenotypes, such as cardiac hypertrophy and survival rate; however, a dose of 300 mg·kg−1 was required to improve symptoms in CSQ‐tg mice. Our results suggest that this newly generated triple‐tg heart failure model is useful to evaluate the cardioprotective effects of human renin inhibitors at clinically relevant doses, thereby minimizing the concerns of off‐target effects related to much higher drug exposure than that achieved in clinical study.
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Affiliation(s)
- Tomoya Hara
- Takeda Pharmaceutical Co Ltd Shonan Research Center, Fujisawa, Japan
| | - Takeshi Yamamura
- Takeda Pharmaceutical Co Ltd Shonan Research Center, Fujisawa, Japan
| | | | | | | | - Ray Kanagawa
- Takeda Pharmaceutical Co Ltd Shonan Research Center, Fujisawa, Japan
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16
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Shen JB, Toti KS, Chakraborty S, Kumar TS, Cronin C, Liang BT, Jacobson KA. Prevention and rescue of cardiac dysfunction by methanocarba adenosine monophosphonate derivatives. Purinergic Signal 2020; 16:61-72. [PMID: 31989534 DOI: 10.1007/s11302-020-09688-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Accepted: 01/15/2020] [Indexed: 11/25/2022] Open
Abstract
Accumulating evidence supports a therapeutic role of purinergic signaling in cardiac diseases. Previously, efficacy of systemically infused MRS2339, a charged methanocarba derivative of 2-Cl-adenosine monophosphate, was demonstrated in animal models of heart failure. We now test the hypothesis that an uncharged adenine nucleoside phosphonate, suitable as an oral agent with a hydrolysis-resistant phospho moiety, can prevent the development of cardiac dysfunction in a post-infarction ischemic or pressure overload-induced heart failure model in mice. The diester-masked uncharged phosphonate MRS2978 was efficacious in preventing cardiac dysfunction with improved left ventricular (LV) fractional shortening when administered orally at the onset of ischemic or pressure overload-induced heart failure. MRS2925, the charged, unmasked MRS2978 analog, prevented heart dysfunction when infused subcutaneously but not by oral gavage. When administered orally or systemically, MRS2978 but not MRS2925 could also rescue established cardiac dysfunction in both ischemic and pressure overload heart failure models. The diester-masked phosphate MRS4074 was highly efficacious at preventing the development of dysfunction as well as in rescuing pressure overload-induced and ischemic heart failure. MRS2978 was orally bioavailable (57-75%) giving rise to MRS2925 as a minor metabolite in vivo, tested in rats. The data are consistent with a novel therapeutic role of adenine nucleoside phosphonates in systolic heart failure.
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Affiliation(s)
- Jian-Bing Shen
- Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT, 06030, USA
| | - Kiran S Toti
- NIDDK, National Institutes of Health, Bethesda, MD, 20892, USA
| | | | | | - Chunxia Cronin
- Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT, 06030, USA
| | - Bruce T Liang
- Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT, 06030, USA.
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17
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Cacheux M, Fauconnier J, Thireau J, Osseni A, Brocard J, Roux-Buisson N, Brocard J, Fauré J, Lacampagne A, Marty I. Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT in the Mouse. Mol Ther 2019; 28:171-179. [PMID: 31607542 DOI: 10.1016/j.ymthe.2019.09.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Revised: 08/28/2019] [Accepted: 09/06/2019] [Indexed: 01/07/2023] Open
Abstract
Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.
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Affiliation(s)
- Marine Cacheux
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Jérémy Fauconnier
- University of Montpellier, INSERM U1046, CNRS 9214, CHU Montpellier, 34295 Montpellier, France
| | - Jérôme Thireau
- University of Montpellier, INSERM U1046, CNRS 9214, CHU Montpellier, 34295 Montpellier, France
| | - Alexis Osseni
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Jacques Brocard
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Nathalie Roux-Buisson
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Julie Brocard
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Julien Fauré
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France
| | - Alain Lacampagne
- University of Montpellier, INSERM U1046, CNRS 9214, CHU Montpellier, 34295 Montpellier, France.
| | - Isabelle Marty
- Grenoble Institut Neurosciences, INSERM, Grenoble Alpes University, U1216, CHU Grenoble Alpes, 38700 La Tronche, France.
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18
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Renin Activity in Heart Failure with Reduced Systolic Function-New Insights. Int J Mol Sci 2019; 20:ijms20133182. [PMID: 31261774 PMCID: PMC6651297 DOI: 10.3390/ijms20133182] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2019] [Revised: 06/26/2019] [Accepted: 06/27/2019] [Indexed: 12/27/2022] Open
Abstract
Regardless of the cause, symptomatic heart failure (HF) with reduced ejection fraction (rEF) is characterized by pathological activation of the renin–angiotensin–aldosterone system (RAAS) with sodium retention and extracellular fluid expansion (edema). Here, we review the role of active renin, a crucial, upstream enzymatic regulator of the RAAS, as a prognostic and diagnostic plasma biomarker of heart failure with reduced ejection fraction (HFrEF) progression; we also discuss its potential as a pharmacological bio-target in HF therapy. Clinical and experimental studies indicate that plasma renin activity is elevated with symptomatic HFrEF with edema in patients, as well as in companion animals and experimental models of HF. Plasma renin activity levels are also reported to be elevated in patients and animals with rEF before the development of symptomatic HF. Modulation of renin activity in experimental HF significantly reduces edema formation and the progression of systolic dysfunction and improves survival. Thus, specific assessment and targeting of elevated renin activity may enhance diagnostic and therapeutic precision to improve outcomes in appropriate patients with HFrEF.
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19
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Lang D, Glukhov AV. Functional Microdomains in Heart's Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling. Front Physiol 2018; 9:1686. [PMID: 30538641 PMCID: PMC6277479 DOI: 10.3389/fphys.2018.01686] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 11/09/2018] [Indexed: 12/12/2022] Open
Abstract
Spontaneous beating of the sinoatrial node (SAN), the primary pacemaker of the heart, is initiated, sustained, and regulated by a complex system that integrates ion channels and transporters on the cell membrane surface (often referred to as "membrane clock") with subcellular calcium handling machinery (by parity of reasoning referred to as an intracellular "Ca2+ clock"). Stable, rhythmic beating of the SAN is ensured by a rigorous synchronization between these two clocks highlighted in the coupled-clock system concept of SAN timekeeping. The emerging results demonstrate that such synchronization of the complex pacemaking machinery at the cellular level depends on tightly regulated spatiotemporal signals which are restricted to precise sub-cellular microdomains and associated with discrete clusters of different ion channels, transporters, and regulatory receptors. It has recently become evident that within the microdomains, various proteins form an interacting network and work together as a part of a macromolecular signaling complex. These protein-protein interactions are tightly controlled and regulated by a variety of neurohormonal signaling pathways and the diversity of cellular responses achieved with a limited pool of second messengers is made possible through the organization of essential signal components in particular microdomains. In this review, we highlight the emerging understanding of the functionality of distinct subcellular microdomains in SAN myocytes and their functional role in the accumulation and neurohormonal regulation of proteins involved in cardiac pacemaking. We also demonstrate how changes in scaffolding proteins may lead to microdomain-targeted remodeling and regulation of pacemaker proteins contributing to SAN dysfunction.
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Affiliation(s)
- Di Lang
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, United States
| | - Alexey V Glukhov
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, United States
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20
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Hara T, Nishimura S, Yamamoto T, Kajimoto Y, Kusumoto K, Kanagawa R, Ikeda S, Nishimoto T. TAK-272 (imarikiren), a novel renin inhibitor, improves cardiac remodeling and mortality in a murine heart failure model. PLoS One 2018; 13:e0202176. [PMID: 30092100 PMCID: PMC6084973 DOI: 10.1371/journal.pone.0202176] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 07/30/2018] [Indexed: 01/19/2023] Open
Abstract
The renin-angiotensin system (RAS), which plays an important role in the progression of heart failure, is efficiently blocked by the inhibition of renin, the rate-limiting enzyme in the RAS cascade. In the present study, we investigated the cardioprotective effects of TAK-272 (SCO-272, imarikiren), a novel, orally effective direct renin inhibitor (DRI), and compared its efficacy with that of aliskiren, a DRI that is already available in the market. TAK-272 was administered to calsequestrin transgenic (CSQ-tg) heart failure mouse model that show severe symptoms and high mortality. The CSQ-tg mice treated with 300 mg/kg, the highest dose tested, of TAK-272 showed significantly reduced plasma renin activity (PRA), cardiac hypertrophy, and lung congestion. Further, TAK-272 reduced cardiomyocyte injury accompanied by an attenuation of the increase in NADPH oxidase 4 and nitric oxide synthase 3 expressions. TAK-272 also prolonged the survival of CSQ-tg mice in a dose-dependent manner (30 mg/kg: P = 0.42, 100 mg/kg: P = 0.12, 300 mg/kg: P < 0.01). Additionally, when compared at the same dose level (300 mg/kg), TAK-272 showed strong and sustained PRA inhibition and reduced the heart weight and plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration, a heart failure biomarker, while aliskiren showed a significant weaker PRA inhibition and failed to demonstrate any cardioprotective effects. Our results showed that TAK-272 is an orally active and persistent renin inhibitor, which reduced the mortality of CSQ-tg mice and conferred protection against cardiac hypertrophy and injury. Thus, TAK-272 treatment could provide a new therapeutic approach for heart failure.
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Affiliation(s)
- Tomoya Hara
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Satoshi Nishimura
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Toshihiro Yamamoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Yumiko Kajimoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Keiji Kusumoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Ray Kanagawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Shota Ikeda
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Tomoyuki Nishimoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
- * E-mail:
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21
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Guo W, Zhu C, Yin Z, Wang Q, Sun M, Cao H, Greaser ML. Splicing Factor RBM20 Regulates Transcriptional Network of Titin Associated and Calcium Handling Genes in The Heart. Int J Biol Sci 2018; 14:369-380. [PMID: 29725258 PMCID: PMC5930469 DOI: 10.7150/ijbs.24117] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2017] [Accepted: 02/27/2018] [Indexed: 01/28/2023] Open
Abstract
RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.
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Affiliation(s)
- Wei Guo
- Animal Science, University of Wyoming, Laramie, WY 82071, USA.,Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | - Chaoqun Zhu
- Animal Science, University of Wyoming, Laramie, WY 82071, USA.,Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | - Zhiyong Yin
- Animal Science, University of Wyoming, Laramie, WY 82071, USA.,Department of Cardiology, Xi Jing Hospital, Fourth Military Medical University, Xi'an 710032, China
| | - Qiurong Wang
- Animal Science, University of Wyoming, Laramie, WY 82071, USA.,Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | - Mingming Sun
- Animal Science, University of Wyoming, Laramie, WY 82071, USA.,Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | - Huojun Cao
- Iowa Institute for Oral Health Research, College of Dentistry.,Craniofacial Anomalies Research Center, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
| | - Marion L Greaser
- Animal Science, University of Wisconsin-Madison, Madison, WI 53705, USA
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22
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Meissner G. The structural basis of ryanodine receptor ion channel function. J Gen Physiol 2017; 149:1065-1089. [PMID: 29122978 PMCID: PMC5715910 DOI: 10.1085/jgp.201711878] [Citation(s) in RCA: 168] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 10/12/2017] [Indexed: 01/25/2023] Open
Abstract
Large-conductance Ca2+ release channels known as ryanodine receptors (RyRs) mediate the release of Ca2+ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ∼12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ∼70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ∼500 amino acids. The remaining ∼4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.
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Affiliation(s)
- Gerhard Meissner
- Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC
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23
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Estrada-Avilés R, Rodríguez G, Zarain-Herzberg A. The cardiac calsequestrin gene transcription is modulated at the promoter by NFAT and MEF-2 transcription factors. PLoS One 2017; 12:e0184724. [PMID: 28886186 PMCID: PMC5590987 DOI: 10.1371/journal.pone.0184724] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Accepted: 08/08/2017] [Indexed: 12/13/2022] Open
Abstract
Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. Previously, we demonstrated that MEF-2 and SRF binding sites within the human CASQ2 gene (hCASQ2) promoter region are functional in neonatal cardiomyocytes. In this work, we investigated if the calcineurin/NFAT pathway regulates hCASQ2 expression in neonatal cardiomyocytes. The inhibition of NFAT dephosphorylation with CsA or INCA-6, reduced both the luciferase activity of hCASQ2 promoter constructs (-3102/+176 bp and -288/+176 bp) and the CASQ2 mRNA levels in neonatal rat cardiomyocytes. Additionally, NFATc1 and NFATc3 over-expressing neonatal cardiomyocytes showed a 2-3-fold increase in luciferase activity of both hCASQ2 promoter constructs, which was prevented by CsA treatment. Site-directed mutagenesis of the -133 bp MEF-2 binding site prevented trans-activation of hCASQ2 promoter constructs induced by NFAT overexpression. Chromatin Immunoprecipitation (ChIP) assays revealed NFAT and MEF-2 enrichment within the -288 bp to +76 bp of the hCASQ2 gene promoter. Besides, a direct interaction between NFAT and MEF-2 proteins was demonstrated by protein co-immunoprecipitation experiments. Taken together, these data demonstrate that NFAT interacts with MEF-2 bound to the -133 bp binding site at the hCASQ2 gene promoter. In conclusion, in this work, we demonstrate that the Ca2+-calcineurin/NFAT pathway modulates the transcription of the hCASQ2 gene in neonatal cardiomyocytes.
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Affiliation(s)
- Rafael Estrada-Avilés
- Department of Biochemistry, School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico
| | - Gabriela Rodríguez
- Department of Biochemistry, School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico
| | - Angel Zarain-Herzberg
- Department of Biochemistry, School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico
- * E-mail:
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24
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Gergs U, Fahrion CM, Bock P, Fischer M, Wache H, Hauptmann S, Schmitz W, Neumann J. Evidence for a functional role of calsequestrin 2 in mouse atrium. Acta Physiol (Oxf) 2017; 219:669-682. [PMID: 27484853 DOI: 10.1111/apha.12766] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 02/23/2016] [Accepted: 07/30/2016] [Indexed: 12/01/2022]
Abstract
AIM Several genetically modified mice models were studied so far to investigate the role of cardiac calsequestrin (CSQ2) for the contractile function of the ventricle and for the occurrence of ventricular tachycardia. Using a CSQ2 knockout mouse, we wanted to study also the atrial function of CSQ2. METHODS The influence of CSQ2 on atrial function and, for comparison, ventricular function was studied in isolated cardiac preparations and by echocardiography as well as electrocardiography in mice with deletion of CSQ2. RESULTS Using deletion of exon 1, we have successfully generated a constitutive knockout mouse of the calsequestrin 2 gene (CSQ2-/- ). CSQ2 protein was absent in the heart (atrium, ventricle), but also in oesophagus and skeletal muscle of homozygous knockout mice. In 6-month-old CSQ2-/- mice, relative left atrial weight was increased, whereas relative heart weight was unchanged. The staircase phenomena in paced left atrial preparations on force of contraction and the post-rest potentiation were different between wild type and CSQ2-/- indicative for a decreased sarcoplasmic Ca2+ load and supporting an important role of CSQ2 also in the atrium. The incidence of arrhythmias was increased in CSQ2-/- . In 2-year-old CSQ2-/- mice, cardiac hypertrophy and heart failure were noted possibly as a result of chronically increased cytosolic Ca2+ levels. CONCLUSION These data suggest a functional role of CSQ2 not only in the ventricle but also in the atrium of mammalian hearts. Loss of CSQ2 function can cause not only arrhythmias, but also cardiac hypertrophy and heart failure.
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Affiliation(s)
- U. Gergs
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
| | - C. M. Fahrion
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
| | - P. Bock
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
| | - M. Fischer
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
| | - H. Wache
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
| | - S. Hauptmann
- Institut für Pathologie am Krankenhaus Düren gGmbH; Düren Germany
| | - W. Schmitz
- Institut für Pharmakologie und Toxikologie; Universitätsklinikum Münster; Münster Germany
| | - J. Neumann
- Institut für Pharmakologie und Toxikologie; Medizinische Fakultät; Martin-Luther-Universität Halle-Wittenberg; Halle/Saale Germany
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25
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Kaneko M, Hashikami K, Yamamoto S, Matsumoto H, Nishimoto T. Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model. PLoS One 2016; 11:e0168486. [PMID: 27992596 PMCID: PMC5161475 DOI: 10.1371/journal.pone.0168486] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Accepted: 11/30/2016] [Indexed: 01/18/2023] Open
Abstract
Sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and its inhibitory protein called phospholamban (PLN) are pivotal for Ca2+ handling in cardiomyocyte and are known that their expression level and activity were changed in the heart failure patients. To examine whether PLN inhibition can improve survival rate as well as cardiac function in heart failure, we performed PLN ablation in calsequestrin overexpressing (CSQ-Tg) mice, a severe heart failure model, using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system. According this method, generation rate of PLN wild type mice (PLN copy >0.95) and PLN homozygous knockout (KO) mice (PLN copy <0.05) were 39.1% and 10.5%, respectively. While CSQ overexpression causes severe heart failure symptoms and premature death, a significant ameliorating effect on survival rate was observed in PLN homozygous KO/CSQ-Tg mice compared to PLN wild type/CSQ-Tg mice (median survival days are 55 and 50 days, respectively). Measurement of cardiac function with cardiac catheterization at the age of 5 weeks revealed that PLN ablation improved cardiac function in CSQ-Tg mice without affecting heart rate and blood pressure. Furthermore, increases in atrial and lung weight, an index of congestion, were significantly inhibited by PLN ablation. These results suggest that PLN deletion would be a promising approach to improve both mortality and cardiac function in the heart failure.
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Affiliation(s)
- Manami Kaneko
- Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
- * E-mail:
| | - Kentarou Hashikami
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Satoshi Yamamoto
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Hirokazu Matsumoto
- Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
| | - Tomoyuki Nishimoto
- Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan
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26
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Handhle A, Ormonde CE, Thomas NL, Bralesford C, Williams AJ, Lai FA, Zissimopoulos S. Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain. J Cell Sci 2016; 129:3983-3988. [PMID: 27609834 PMCID: PMC5117208 DOI: 10.1242/jcs.191643] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2016] [Accepted: 09/05/2016] [Indexed: 11/20/2022] Open
Abstract
Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.
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Affiliation(s)
- Ahmed Handhle
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.,Medical Biochemistry Department, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt
| | - Chloe E Ormonde
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
| | - N Lowri Thomas
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
| | - Catherine Bralesford
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
| | - Alan J Williams
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
| | - F Anthony Lai
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
| | - Spyros Zissimopoulos
- Sir Geraint Evans Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
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27
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Kaneko M, Satomi T, Fujiwara S, Uchiyama H, Kusumoto K, Nishimoto T. AT1 receptor blocker azilsartan medoxomil normalizes plasma miR-146a and miR-342-3p in a murine heart failure model. Biomarkers 2016; 22:253-260. [PMID: 27321284 DOI: 10.1080/1354750x.2016.1204001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Our study measured circulating microRNA (miRNA) levels in the plasma of calsequestrin (CSQ)-tg mouse, a severe heart failure model, and evaluated whether treatment with angiotensin II type 1 receptor blocker, azilsartan medoxomil (AZL-M) influenced their levels using miRNA array analysis. MiR-146a, miR-149, miR-150, and miR-342-3p were reproducibly reduced in the plasma of CSQ-tg mice. Among them, miR-146a and miR-342-3p were significantly restored by AZL-M, which were associated with improvement of survival rate and reduction of congestion. These results suggest that miRNA, especially miR-146a and miR-342-3p, could be used as potential biomarkers for evaluating the efficacy of anti-heart failure drugs.
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Affiliation(s)
- Manami Kaneko
- a Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
| | - Tomoko Satomi
- a Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
| | - Shuji Fujiwara
- a Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
| | - Hidefumi Uchiyama
- b Integrated Technology Research Laboratories, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
| | - Keiji Kusumoto
- a Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
| | - Tomoyuki Nishimoto
- a Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division , Takeda Pharmaceutical Company Limited , Fujisawa , Japan
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28
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Guizoni DM, Oliveira-Junior SA, Noor SLR, Pagan LU, Martinez PF, Lima ARR, Gomes MJ, Damatto RL, Cezar MDM, Bonomo C, Zornoff LAM, Okoshi K, Okoshi MP. Effects of late exercise on cardiac remodeling and myocardial calcium handling proteins in rats with moderate and large size myocardial infarction. Int J Cardiol 2016; 221:406-12. [PMID: 27404715 DOI: 10.1016/j.ijcard.2016.07.072] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2016] [Accepted: 07/04/2016] [Indexed: 01/10/2023]
Abstract
BACKGROUND Physical exercise attenuates myocardial infarction (MI)-induced cardiac remodeling. However, it is unsettled whether late exercise modulates post-infarction cardiac remodeling differentially according to infarct size. We investigated the effects of exercise started at late stage heart failure on cardiac remodeling in rats with moderate and large sized MI. METHODS Three months after MI, rats were assigned into sedentary and exercise groups. Exercise rats underwent treadmill for three months. After assessing infarct size by histological analysis, rats were subdivided into four groups: moderate MI sedentary (Mod MI-Sed; n=7), Mod MI exercised (Mod MI-Ex; n=7), Large MI-Sed (n=11), and Large MI-Ex (n=10). RESULTS Before exercise, MI-induced cardiac changes were demonstrated by comparing results to a Sham group; alterations were more intense in rats with large than moderate MI size. Systolic function, evaluated by echocardiogram using the variation in LV fractional area change between after and before exercise, was improved in exercise than sedentary groups. Calsequestrin expression increased in exercised compared to sedentary groups. L-type calcium channel was higher in Mod MI-Ex than Mod MI-Sed. SERCA2a, phospholamban, and Na(+)/Ca(2+) exchanger expression did not differ between groups. CONCLUSION Late exercise improves systolic function and modulates intracellular calcium signaling proteins in rats with moderate and large MI.
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Affiliation(s)
- Daniele M Guizoni
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | | | - Sefora L R Noor
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Luana U Pagan
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Paula F Martinez
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil; School of Physical Therapy, Federal University of Mato Grosso do Sul, Campo Grande, Brazil
| | - Aline R R Lima
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Mariana J Gomes
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Ricardo L Damatto
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Marcelo D M Cezar
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Camila Bonomo
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Leonardo A M Zornoff
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Katashi Okoshi
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil
| | - Marina P Okoshi
- Internal Medicine Department, Botucatu Medical School, Sao Paulo State University, UNESP, Botucatu, Brazil.
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29
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Molinari F, Malara N, Mollace V, Rosano G, Ferraro E. Animal models of cardiac cachexia. Int J Cardiol 2016; 219:105-10. [PMID: 27317993 DOI: 10.1016/j.ijcard.2016.05.071] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Accepted: 05/29/2016] [Indexed: 02/06/2023]
Abstract
Cachexia is the loss of body weight associated with several chronic diseases including chronic heart failure (CHF). The cachectic condition is mainly due to loss of skeletal muscle mass and adipose tissue depletion. The majority of experimental in vivo studies on cachexia rely on animal models of cancer cachexia while a reliable and appropriate model for cardiac cachexia has not yet been established. A critical issue in generating a cardiac cachexia model is that genetic modifications or pharmacological treatments impairing the heart functionality and used to obtain the heart failure model might likely impair the skeletal muscle, this also being a striated muscle and sharing with the myocardium several molecular and physiological mechanisms. On the other hand, often, the induction of heart damage in the several existing models of heart failure does not necessarily lead to skeletal muscle loss and cachexia. Here we describe the main features of cardiac cachexia and illustrate some animal models proposed for cardiac cachexia studies; they include the genetic calsequestrin and Dahl salt-sensitive models, the monocrotaline model and the surgical models obtained by left anterior descending (LAD) ligation, transverse aortic constriction (TAC) and ascending aortic banding. The availability of a specific animal model for cardiac cachexia is a crucial issue since, besides the common aspects of cachexia in the different syndromes, each disease has some peculiarities in its etiology and pathophysiology leading to cachexia. Such peculiarities need to be unraveled in order to find new targets for effective therapies.
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Affiliation(s)
- Francesca Molinari
- Laboratory of Pathophysiology of Cachexia and Metabolism of Skeletal Muscle, IRCCS San Raffaele Pisana, Rome, Italy
| | - Natalia Malara
- Interregional Research Center on Food Safety & Health (IRC-FSH), Department of Health Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy
| | - Vincenzo Mollace
- Interregional Research Center on Food Safety & Health (IRC-FSH), Department of Health Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy
| | - Giuseppe Rosano
- Laboratory of Pathophysiology of Cachexia and Metabolism of Skeletal Muscle, IRCCS San Raffaele Pisana, Rome, Italy; Cardiovascular and Cell Sciences Institute, St George's University of London, Cranmer Terrace, London, UK
| | - Elisabetta Ferraro
- Laboratory of Pathophysiology of Cachexia and Metabolism of Skeletal Muscle, IRCCS San Raffaele Pisana, Rome, Italy.
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30
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Ríos E, Figueroa L, Manno C, Kraeva N, Riazi S. The couplonopathies: A comparative approach to a class of diseases of skeletal and cardiac muscle. ACTA ACUST UNITED AC 2016; 145:459-74. [PMID: 26009541 PMCID: PMC4442791 DOI: 10.1085/jgp.201411321] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
A novel category of diseases of striated muscle is proposed, the couplonopathies, as those that affect components of the couplon and thereby alter its operation. Couplons are the functional units of intracellular calcium release in excitation–contraction coupling. They comprise dihydropyridine receptors, ryanodine receptors (Ca2+ release channels), and a growing list of ancillary proteins whose alteration may lead to disease. Within a generally similar plan, the couplons of skeletal and cardiac muscle show, in a few places, marked structural divergence associated with critical differences in the mechanisms whereby they fulfill their signaling role. Most important among these are the presence of a mechanical or allosteric communication between voltage sensors and Ca2+ release channels, exclusive to the skeletal couplon, and the smaller capacity of the Ca stores in cardiac muscle, which results in greater swings of store concentration during physiological function. Consideration of these structural and functional differences affords insights into the pathogenesis of several couplonopathies. The exclusive mechanical connection of the skeletal couplon explains differences in pathogenesis between malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT), conditions most commonly caused by mutations in homologous regions of the skeletal and cardiac Ca2+ release channels. Based on mechanistic considerations applicable to both couplons, we identify the plasmalemma as a site of secondary modifications, typically an increase in store-operated calcium entry, that are relevant in MH pathogenesis. Similar considerations help explain the different consequences that mutations in triadin and calsequestrin have in these two tissues. As more information is gathered on the composition of cardiac and skeletal couplons, this comparative and mechanistic approach to couplonopathies should be useful to understand pathogenesis, clarify diagnosis, and propose tissue-specific drug development.
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Affiliation(s)
- Eduardo Ríos
- Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612
| | - Lourdes Figueroa
- Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612
| | - Carlo Manno
- Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612
| | - Natalia Kraeva
- Malignant Hyperthermia Investigation Unit, University Health Network, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada
| | - Sheila Riazi
- Malignant Hyperthermia Investigation Unit, University Health Network, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada
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31
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Zungu-Edmondson M, Shults NV, Wong CM, Suzuki YJ. Modulators of right ventricular apoptosis and contractility in a rat model of pulmonary hypertension. Cardiovasc Res 2016; 110:30-9. [PMID: 26790474 DOI: 10.1093/cvr/cvw014] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 12/30/2015] [Indexed: 11/12/2022] Open
Abstract
AIMS Right ventricular (RV) failure is the major cause of death among patients with pulmonary arterial hypertension (PAH). However, the mechanism of RV failure has not been defined. METHODS AND RESULTS This study examined mechanisms and consequences of RV myocyte apoptosis and fibrosis in response to PAH. Rats were injected with SU5416 (vascular endothelial growth factor inhibitor), followed by hypoxia for 3 weeks, and subsequently maintained in normoxia for 2, 5, or 14 weeks (5-, 8-, and 17-week time points after the SU5416 injection, respectively). RV systolic pressure (RVSP) was elevated to >70 mmHg at 5-week time point, and this pressure was sustained thereafter. Significant RV myocyte apoptosis and fibrosis were observed at 8- and 17-week time points. Apoptosis was associated with downregulated Bcl-xL (anti-apoptotic protein), downregulated GATA4 (transcriptional regulator of Bcl-xL), and upregulated p53 (negative regulator of GATA4 gene transcription). PAH-mediated RV apoptosis and fibrosis were attenuated in p53 knock-out rats. Despite the major loss of cardiomyocytes, RV contractility was enhanced, suggesting that the remaining myocytes can perform improved contractile functions. Improved RV contractility is associated with the increased expression of contractile and sarcoplasmic reticulum Ca(2+) uptake proteins. In contrast, the expression of calsequestrin 2 (CSQ2) was downregulated. The siRNA knockdown of CSQ2 improved RV contractility and increased the expression of contractile and Ca(2+) uptake proteins. CONCLUSION These results suggest that RV decompensation is associated with the death of cardiomyocytes, resulting in fibrosis. However, the remaining myocytes are capable of sustaining RV contractility through the mechanism that involves CSQ2.
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Affiliation(s)
- Makhosazane Zungu-Edmondson
- Department of Pharmacology and Physiology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20057, USA
| | - Nataliia V Shults
- Department of Pharmacology and Physiology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20057, USA
| | - Chi-Ming Wong
- Department of Pharmacology and Physiology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20057, USA
| | - Yuichiro J Suzuki
- Department of Pharmacology and Physiology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20057, USA
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Di Blasi C, Sansanelli S, Ruggieri A, Moriggi M, Vasso M, D'Adamo AP, Blasevich F, Zanotti S, Paolini C, Protasi F, Tezzon F, Gelfi C, Morandi L, Pessia M, Mora M. A CASQ1 founder mutation in three Italian families with protein aggregate myopathy and hyperCKaemia. J Med Genet 2015; 52:617-26. [PMID: 26136523 DOI: 10.1136/jmedgenet-2014-102882] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 06/16/2015] [Indexed: 12/24/2022]
Abstract
BACKGROUND Protein aggregate myopathies are increasingly recognised conditions characterised by a surplus of endogenous proteins. The molecular and mutational background for many protein aggregate myopathies has been clarified with the discovery of several underlying mutations. Familial idiopathic hyperCKaemia is a benign genetically heterogeneous condition with autosomal dominant features in a high proportion of cases. METHODS In 10 patients from three Italian families with autosomal dominant benign vacuolar myopathy and hyperCKaemia, we performed linkage analysis and exome sequencing as well as morphological and biochemical investigations. RESULTS AND CONCLUSIONS We show, by Sanger and exome sequencing, that the protein aggregate myopathy with benign evolution and muscle inclusions composed of excess CASQ1, affecting three Italian families, is due to the D244G heterozygous missense mutation in the CASQ1 gene. Investigation of microsatellite markers revealed a common haplotype in the three families indicating consanguinity and a founder effect. Results from immunocytochemistry, electron microscopy, biochemistry and transfected cell line investigations contribute to our understanding of pathogenetic mechanisms underlining this defect. The mutation is common to other Italian patients and is likely to share a founder effect with them. HyperCKaemia in the CASQ1-related myopathy is common and sometimes the sole overt manifestation. It is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed, and the condition could be more common than supposed.
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Affiliation(s)
- Claudia Di Blasi
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Serena Sansanelli
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Alessandra Ruggieri
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Manuela Moriggi
- Department of Biomedical Sciences for Health, University of Milano, Milano, Italy
| | - Michele Vasso
- Department of Biomedical Sciences for Health, University of Milano, Milano, Italy CNR-Institute of Bioimaging and Molecular Physiology, Milano, Italy
| | | | - Flavia Blasevich
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Simona Zanotti
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Cecilia Paolini
- CeSI, Center for Research on Ageing & Department of Neuroscience, Imaging, and Clinical Sciences, University G D'Annunzio of Chieti, Chieti, Italy
| | - Feliciano Protasi
- CeSI, Center for Research on Ageing & Department of Neuroscience, Imaging, and Clinical Sciences, University G D'Annunzio of Chieti, Chieti, Italy
| | - Frediano Tezzon
- Neurology Unit, F Tappeiner Hospital of Merano, Merano, Italy
| | - Cecilia Gelfi
- Department of Biomedical Sciences for Health, University of Milano, Milano, Italy CNR-Institute of Bioimaging and Molecular Physiology, Milano, Italy
| | - Lucia Morandi
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
| | - Mauro Pessia
- Faculty of Medicine, Section of Physiology and Biochemistry, Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Marina Mora
- Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy
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Paolini C, Quarta M, Wei-LaPierre L, Michelucci A, Nori A, Reggiani C, Dirksen RT, Protasi F. Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice. Skelet Muscle 2015; 5:10. [PMID: 26075051 PMCID: PMC4464246 DOI: 10.1186/s13395-015-0035-9] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Accepted: 03/19/2015] [Indexed: 12/17/2022] Open
Abstract
Background Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans. Methods We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays. Results About 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice. Conclusions Our findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function. Electronic supplementary material The online version of this article (doi:10.1186/s13395-015-0035-9) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Cecilia Paolini
- CeSI - Center for Research on Ageing & DNICS - Department of Neuroscience, Imaging and Clinical Sciences, University G. d'Annunzio, Via L. Polacchi, 11, I-66013 Chieti, Italy
| | - Marco Quarta
- Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy ; Department of Neurology and Neurological Sciences, Stanford University, 450 Serra Mall, Stanford, CA 94305 USA
| | - Lan Wei-LaPierre
- Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 USA
| | - Antonio Michelucci
- CeSI - Center for Research on Ageing & DNICS - Department of Neuroscience, Imaging and Clinical Sciences, University G. d'Annunzio, Via L. Polacchi, 11, I-66013 Chieti, Italy
| | - Alessandra Nori
- Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy
| | - Carlo Reggiani
- Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy
| | - Robert T Dirksen
- Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 USA
| | - Feliciano Protasi
- CeSI - Center for Research on Ageing & DNICS - Department of Neuroscience, Imaging and Clinical Sciences, University G. d'Annunzio, Via L. Polacchi, 11, I-66013 Chieti, Italy
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34
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Zhang JZ, Waddell HMM, Jones PP. Regulation of RYR2 by sarcoplasmic reticulum Ca(2+). Clin Exp Pharmacol Physiol 2015; 42:720-6. [PMID: 25603835 DOI: 10.1111/1440-1681.12364] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2014] [Revised: 09/17/2014] [Accepted: 10/09/2014] [Indexed: 11/28/2022]
Abstract
Ca(2+) is arguably the most important ion involved in the contraction of the heart. The cardiac ryanodine receptor (RyR2), the major Ca(2+) release channel located in the sarcoplasmic reticulum (SR) membrane, is responsible for releasing the bulk of Ca(2+) required for contraction. Moreover, RyR2 is also crucial for maintaining SR Ca(2+) homeostasis by releasing Ca(2+) from the SR when it becomes overloaded with Ca(2+) . During normal contraction, RyR2 is activated by cytosolic Ca(2+) , whereas during store overload conditions, the opening of RyR2 is governed by SR Ca(2+) . Although the process of the cytosolic control of RyR2 is well established, the molecular mechanism by which SR luminal Ca(2+) regulates RyR2 has only recently been elucidated and remains controversial. In addition to the activation of RyR2, SR luminal Ca(2+) also determines when the RyR2 channel closes. RyR2-mediated Ca(2+) release from the SR does not continue until the SR is completely depleted. Rather, it ceases when SR luminal Ca(2+) falls below a certain level. Given the importance of SR Ca(2+) , it is not surprising that the SR luminal Ca(2+) level is tightly controlled by SR Ca(2+) -buffering proteins. Consequently, the opening and closing of RyR2 is heavily influenced by the presence of such proteins, particularly those associated with RyR2, such as calsequestrin and the histidine-rich Ca(2+) -binding protein. These proteins appear to indirectly alter RyR2 activity by modifying the microdomain SR Ca(2+) level surrounding RyR2.
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Affiliation(s)
- Joe Z Zhang
- Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
| | - Helen M M Waddell
- Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
| | - Peter P Jones
- Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
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35
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Song W, Wang X. The role of TGFβ1 and LRG1 in cardiac remodelling and heart failure. Biophys Rev 2015; 7:91-104. [PMID: 28509980 PMCID: PMC4322186 DOI: 10.1007/s12551-014-0158-y] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2014] [Accepted: 11/26/2014] [Indexed: 12/12/2022] Open
Abstract
Heart failure is a life-threatening condition that carries a considerable emotional and socio-economic burden. As a result of the global increase in the ageing population, sedentary life-style, increased prevalence of risk factors, and improved survival from cardiovascular events, the incidence of heart failure will continue to rise. Despite the advances in current cardiovascular therapies, many patients are not suitable for or may not benefit from conventional treatments. Thus, more effective therapies are required. Transforming growth factor (TGF) β family of cytokines is involved in heart development and dys-regulated TGFβ signalling is commonly associated with fibrosis, aberrant angiogenesis and accelerated progression into heart failure. Therefore, a potential therapeutic pathway is to modulate TGFβ signalling; however, broad blockage of TGFβ signalling may cause unwanted side effects due to its pivotal role in tissue homeostasis. We found that leucine-rich α-2 glycoprotein 1 (LRG1) promotes blood vessel formation via regulating the context-dependent endothelial TGFβ signalling. This review will focus on the interaction between LRG1 and TGFβ signalling, their involvement in the pathogenesis of heart failure, and the potential for LRG1 to function as a novel therapeutic target.
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Affiliation(s)
- Weihua Song
- Division of Metabolic Medicine, Lee Kong Chian School of Medicine, Nanyang Technological University, Research Techno Plaza, X-Frontiers Block, Level 4, 50 Nan yang Drive, Singapore, 637553, Singapore
| | - Xiaomeng Wang
- Division of Metabolic Medicine, Lee Kong Chian School of Medicine, Nanyang Technological University, Research Techno Plaza, X-Frontiers Block, Level 4, 50 Nan yang Drive, Singapore, 637553, Singapore. .,Division of Cell Biology in Health and Disease, Institute of Molecular and Cell Biology, Singapore Agency for Science, Technology and Research, 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore. .,Department of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.
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36
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Locatelli J, de Assis LVM, Isoldi MC. Calcium handling proteins: structure, function, and modulation by exercise. Heart Fail Rev 2014; 19:207-25. [PMID: 23436107 DOI: 10.1007/s10741-013-9373-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Heart failure is a serious public health issue with a growing prevalence, and it is related with the aging of the population. Hypertension is identified as the main precursor of left ventricular hypertrophy and therefore can lead to diastolic dysfunction and heart failure. Scientific studies have confirmed the beneficial effects of the physical exercise by reducing the blood pressure and improving the functional status of the heart in hypertension. Several proteins are involved in the mobilization of calcium during the coupling excitation-contraction process in the heart among those are sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban, calsequestrin, sodium-calcium exchanger, L-type calcium's channel, and ryanodine receptors. Our goal is to address the beneficial effects of exercise on the calcium handling proteins in a heart with hypertension.
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Affiliation(s)
- Jamille Locatelli
- Institute of Exact and Biological Sciences, Federal University of Ouro Preto, Ouro Prêto, Brazil
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Rossi D, Vezzani B, Galli L, Paolini C, Toniolo L, Pierantozzi E, Spinozzi S, Barone V, Pegoraro E, Bello L, Cenacchi G, Vattemi G, Tomelleri G, Ricci G, Siciliano G, Protasi F, Reggiani C, Sorrentino V. A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates. Hum Mutat 2014; 35:1163-70. [PMID: 25116801 DOI: 10.1002/humu.22631] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2014] [Accepted: 08/05/2014] [Indexed: 12/20/2022]
Abstract
A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.
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Affiliation(s)
- Daniela Rossi
- Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena and Azienda Ospedaliera Universitaria Senese, Siena, 53100, Italy; IIM, Interuniversity Institute of Myology
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Okutsu M, Call JA, Lira VA, Zhang M, Donet JA, French BA, Martin KS, Peirce-Cottler SM, Rembold CM, Annex BH, Yan Z. Extracellular superoxide dismutase ameliorates skeletal muscle abnormalities, cachexia, and exercise intolerance in mice with congestive heart failure. Circ Heart Fail 2014; 7:519-30. [PMID: 24523418 DOI: 10.1161/circheartfailure.113.000841] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
BACKGROUND Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. METHODS AND RESULTS We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (α-myosin heavy chain-calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle. CONCLUSIONS EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF.
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Affiliation(s)
- Mitsuharu Okutsu
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Jarrod A Call
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Vitor A Lira
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Mei Zhang
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Jean A Donet
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Brent A French
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Kyle S Martin
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Shayn M Peirce-Cottler
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Christopher M Rembold
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Brian H Annex
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.)
| | - Zhen Yan
- From the Departments of Medicine (M.O., J.A.C., V.A.L., M.Z., J.A.D., C.M.R., B.H.A., Z.Y.), Pharmacology (Z.Y.), and Molecular Physiology and Biological Physics (Z.Y.), Center for Skeletal Muscle Research (M.O., J.A.C., V.A.L., M.Z., J.A.D., Z.Y.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA; and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA (B.A.F., K.S.M., S.M.P.-C.).
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Fiedler LR, Maifoshie E, Schneider MD. Mouse models of heart failure: cell signaling and cell survival. Curr Top Dev Biol 2014; 109:171-247. [PMID: 24947238 DOI: 10.1016/b978-0-12-397920-9.00002-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Heart failure is one of the paramount global causes of morbidity and mortality. Despite this pandemic need, the available clinical counter-measures have not altered substantially in recent decades, most notably in the context of pharmacological interventions. Cell death plays a causal role in heart failure, and its inhibition poses a promising approach that has not been thoroughly explored. In previous approaches to target discovery, clinical failures have reflected a deficiency in mechanistic understanding, and in some instances, failure to systematically translate laboratory findings toward the clinic. Here, we review diverse mouse models of heart failure, with an emphasis on those that identify potential targets for pharmacological inhibition of cell death, and on how their translation into effective therapies might be improved in the future.
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Affiliation(s)
- Lorna R Fiedler
- British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London, UK.
| | - Evie Maifoshie
- British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London, UK
| | - Michael D Schneider
- British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London, UK.
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The therapeutic effect of 2-cyclohexylthio-AMP in heart failure. J Cardiovasc Pharmacol 2013; 61:553-9. [PMID: 23474842 DOI: 10.1097/fjc.0b013e31828e8758] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
AIM : The aim of this study was to investigate the therapeutic effect of 2-cyclohexylthio-adenosine 5'-monophosphate (AMP) in mice with heart failure (HF). METHODS : 2-Cyclohexylthio-AMP was dissolved in phosphate-buffered saline and infused in mice with ischemic HF after permanent left coronary [left anterior descending (LAD)] ligation and in calsequestrin (CSQ) mice with HF. Myocardial function ex vivo was determined in the working heart model. Cardiac function in vivo was assessed by echocardiography. RESULTS : Injection of 2-cyclohexylthio-AMP induced a dose-dependent increase in +dP/dt, -dP/dt, and left ventricular developed pressure in normal wild-type mice and in CSQ mice with HF using the ex vivo working heart model. Spontaneous heart rate did not change after the injection of 2-cyclohexylthio-AMP. Compared with normal saline-treaded mice, chronic infusion of 2-cyclohexylthio-AMP in mice with ischemic HF after left coronary artery (LAD) ligation and in CSQ mice resulted in improved +dP/dt, -dP/dt, left ventricular developed pressure, and fractional shortening, restored the β-adrenergic response and decreased heart weight/body weight ratios. CONCLUSIONS : 2-Cyclohexylthio-AMP improved the cardiac contractile performance and rescued mice from HF. This salutary action may result from the reduction of myocardial hypertrophy and the restoration of the β-adrenergic response in both LAD ligation and CSQ mouse models of HF. The fact that this agent can increase contractile performance without heart rate increase should be desirable in HF therapy.
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Gomes AC, Falcão-Pires I, Pires AL, Brás-Silva C, Leite-Moreira AF. Rodent models of heart failure: an updated review. Heart Fail Rev 2013; 18:219-49. [PMID: 22446984 DOI: 10.1007/s10741-012-9305-3] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Heart failure (HF) is one of the major health and economic burdens worldwide, and its prevalence is continuously increasing. The study of HF requires reliable animal models to study the chronic changes and pharmacologic interventions in myocardial structure and function and to follow its progression toward HF. Indeed, during the past 40 years, basic and translational scientists have used small animal models to understand the pathophysiology of HF and find more efficient ways of preventing and managing patients suffering from congestive HF (CHF). Each species and each animal model has advantages and disadvantages, and the choice of one model over another should take them into account for a good experimental design. The aim of this review is to describe and highlight the advantages and drawbacks of some commonly used HF rodents models, including both non-genetically and genetically engineered models, with a specific subchapter concerning diastolic HF models.
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Affiliation(s)
- A C Gomes
- Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319, Porto, Portugal
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Gaburjakova M, Bal NC, Gaburjakova J, Periasamy M. Functional interaction between calsequestrin and ryanodine receptor in the heart. Cell Mol Life Sci 2013; 70:2935-45. [PMID: 23109100 PMCID: PMC11113811 DOI: 10.1007/s00018-012-1199-7] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2012] [Revised: 10/02/2012] [Accepted: 10/15/2012] [Indexed: 11/25/2022]
Abstract
Evidence obtained in the last two decades indicates that calsequestrin (CSQ2), as the major Ca(2+)-binding protein in the sarcoplasmic reticulum of cardiac myocytes, communicates changes in the luminal Ca(2+) concentration to the cardiac ryanodine receptor (RYR2) channel. This review summarizes the major aspects in the interaction between CSQ2 and the RYR2 channel. The single channel properties of RYR2 channels, discussed here in the context of structural changes in CSQ2 after Ca(2+) binding, are particularly important. We focus on five important questions concerning: (1) the method for reliable detection of CSQ2 on the reconstituted RYR2 channel complex; (2) the power of the procedure to strip CSQ2 from the RYR2 channel complex; (3) structural changes in CSQ2 upon binding of Ca(2+) which cause CSQ2 dissociation; (4) the potential role of CSQ2-independent regulation of the RYR2 activity by luminal Ca(2+); and (5) the vizualization of CSQ2 dissociation from the RYR2 channel complex on the single channel level. We discuss the potential sources of the conflicting experimental results which may aid detailed understanding of the CSQ2 regulatory role. Although we mainly focus on the cardiac isoform of the proteins, some aspects of more extensive work carried out on the skeletal isoform are also discussed.
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Affiliation(s)
- Marta Gaburjakova
- Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, Bratislava, Slovak Republic.
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Arnáiz-Cot JJ, Damon BJ, Zhang XH, Cleemann L, Yamaguchi N, Meissner G, Morad M. Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor. J Physiol 2013; 591:4287-99. [PMID: 23836685 DOI: 10.1113/jphysiol.2013.256123] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.
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Affiliation(s)
- Juan José Arnáiz-Cot
- M. Morad: Cardiac Signaling Center, 173 Ashley Ave, Bioengineering Building, Room 306, Charleston, SC 29403, USA.
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Mullins PD, Bondarenko VE. A mathematical model of the mouse ventricular myocyte contraction. PLoS One 2013; 8:e63141. [PMID: 23671664 PMCID: PMC3650013 DOI: 10.1371/journal.pone.0063141] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2012] [Accepted: 03/29/2013] [Indexed: 12/05/2022] Open
Abstract
Mathematical models of cardiac function at the cellular level include three major components, such as electrical activity, Ca2+ dynamics, and cellular shortening. We developed a model for mouse ventricular myocyte contraction which is based on our previously published comprehensive models of action potential and Ca2+ handling mechanisms. The model was verified with extensive experimental data on mouse myocyte contraction at room temperature. In the model, we implemented variable sarcomere length and indirect modulation of the tropomyosin transition rates by Ca2+ and troponin. The resulting model described well steady-state force-calcium relationships, dependence of the contraction force on the sarcomere length, time course of the contraction force and myocyte shortening, frequency dependence of the contraction force and cellular contraction, and experimentally measured derivatives of the myocyte length variation. We emphasized the importance of the inclusion of variable sarcomere length into a model for ventricular myocyte contraction. Differences in contraction force and cell shortening for epicardial and endocardial ventricular myocytes were investigated. Model applicability for the experimental studies and model limitations were discussed.
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Affiliation(s)
- Paula D. Mullins
- Department of Mathematics and Statistics, Georgia State University, Atlanta, Georgia, United States of America
| | - Vladimir E. Bondarenko
- Department of Mathematics and Statistics, Georgia State University, Atlanta, Georgia, United States of America
- Neuroscience Institute, Georgia State University, Atlanta, Georgia, United States of America
- * E-mail:
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45
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Waters SB, Diak DM, Zuckermann M, Goldspink PH, Leoni L, Roman BB. Genetic background influences adaptation to cardiac hypertrophy and Ca(2+) handling gene expression. Front Physiol 2013; 4:11. [PMID: 23508205 PMCID: PMC3589715 DOI: 10.3389/fphys.2013.00011] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2012] [Accepted: 01/09/2013] [Indexed: 11/13/2022] Open
Abstract
Genetic variability has a profound effect on the development of cardiac hypertrophy in response to stress. Consequently, using a variety of inbred mouse strains with known genetic profiles may be powerful models for studying the response to cardiovascular stress. To explore this approach we looked at male C57BL/6J and 129/SvJ mice. Hemodynamic analyses of left ventricular pressures (LVPs) indicated significant differences in 129/SvJ and C57BL/6J mice that implied altered Ca(2+) handling. Specifically, 129/SvJ mice demonstrated reduced rates of relaxation and insensitivity to dobutamine (Db). We hypothesized that altered expression of genes controlling the influx and efflux of Ca(2+) from the sarcoplasmic reticulum (SR) was responsible and investigated the expression of several genes involved in maintaining the intracellular and sarcoluminal Ca(2+) concentration using quantitative real-time PCR analyses (qRT-PCR). We observed significant differences in baseline gene expression as well as different responses in expression to isoproterenol (ISO) challenge. In untreated control animals, 129/SvJ mice expressed 1.68× more ryanodine receptor 2(Ryr2) mRNA than C57BL/6J mice but only 0.37× as much calsequestrin 2 (Casq2). After treatment with ISO, sarco(endo)plasmic reticulum Ca(2+)-ATPase(Serca2) expression was reduced nearly two-fold in 129/SvJ while expression in C57BL/6J was stable. Interestingly, β (1) adrenergic receptor(Adrb1) expression was lower in 129/SvJ compared to C57BL/6J at baseline and lower in both strains after treatment. Metabolically, the brain isoform of creatine kinase (Ckb) was up-regulated in response to ISO in C57BL/6J but not in 129/SvJ. These data suggest that the two strains of mice regulate Ca(2+) homeostasis via different mechanisms and may be useful in developing personalized therapies in human patients.
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Affiliation(s)
- Steve B Waters
- Department of Radiology, The University of Chicago Chicago, IL, USA
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Amoasii L, Hnia K, Chicanne G, Brech A, Cowling BS, Müller MM, Schwab Y, Koebel P, Ferry A, Payrastre B, Laporte J. Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo. J Cell Sci 2013; 126:1806-19. [PMID: 23444364 DOI: 10.1242/jcs.118505] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.
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Affiliation(s)
- Leonela Amoasii
- Department of Translational Medicine, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, Université de Strasbourg, Collège de France, 67404 Illkirch, France
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Landstrom AP, Ackerman MJ. Beyond the cardiac myofilament: hypertrophic cardiomyopathy- associated mutations in genes that encode calcium-handling proteins. Curr Mol Med 2012; 12:507-18. [PMID: 22515980 DOI: 10.2174/156652412800620020] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2011] [Revised: 12/30/2011] [Accepted: 01/11/2012] [Indexed: 12/30/2022]
Abstract
Traditionally regarded as a genetic disease of the cardiac sarcomere, hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease and a significant cause of sudden cardiac death. While the most common etiologies of this phenotypically diverse disease lie in a handful of genes encoding critical contractile myofilament proteins, approximately 50% of patients diagnosed with HCM worldwide do not host sarcomeric gene mutations. Recently, mutations in genes encoding calcium-sensitive and calcium-handling proteins have been implicated in the pathogenesis of HCM. Among these are mutations in TNNC1- encoded cardiac troponin C, PLN-encoded phospholamban, and JPH2-encoded junctophilin 2 which have each been associated with HCM in multiple studies. In addition, mutations in RYR2-encoded ryanodine receptor 2, CASQ2-encoded calsequestrin 2, CALR3-encoded calreticulin 3, and SRI-encoded sorcin have been associated with HCM, although more studies are required to validate initial findings. While a relatively uncommon cause of HCM, mutations in genes that encode calcium-handling proteins represent an emerging genetic subset of HCM. Furthermore, these naturally occurring disease-associated mutations have provided useful molecular tools for uncovering novel mechanisms of disease pathogenesis, increasing our understanding of basic cardiac physiology, and dissecting important structure-function relationships within these proteins.
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Affiliation(s)
- A P Landstrom
- Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA
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Calsequestrin mutations and catecholaminergic polymorphic ventricular tachycardia. Pediatr Cardiol 2012; 33:959-67. [PMID: 22421959 PMCID: PMC3393815 DOI: 10.1007/s00246-012-0256-1] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2012] [Accepted: 02/16/2012] [Indexed: 10/28/2022]
Abstract
Cardiac calsequestrin (Casq2) is the major Ca2+ binding protein in the sarcoplasmic reticulum, which is the principle Ca2+ storage organelle of cardiac muscle. During the last decade, experimental studies have provided new concepts on the role of Casq2 in the regulation of cardiac muscle Ca2+ handling. Furthermore, mutations in the gene encoding for cardiac calsequestrin, CASQ2, cause a rare but severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). Here, we review the physiology of Casq2 in cardiac Ca2+ handling and discuss pathophysiological mechanisms that lead to CPVT caused by CASQ2 mutations. We also describe the clinical aspects of CPVT and provide an update of its contemporary clinical management.
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Lorenzi V, Mehinto AC, Denslow ND, Schlenk D. Effects of exposure to the β-blocker propranolol on the reproductive behavior and gene expression of the fathead minnow, Pimephales promelas. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2012; 116-117:8-15. [PMID: 22465857 DOI: 10.1016/j.aquatox.2012.03.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/06/2011] [Revised: 02/28/2012] [Accepted: 03/01/2012] [Indexed: 05/22/2023]
Abstract
Human pharmaceutical drugs have been found in surface waters worldwide, and represent an increasing concern since little is known about their possible effects on wildlife. Propranolol is a common beta-adrenergic receptor antagonist (β-blocker) typically prescribed to people suffering from heart disease and hypertension. Propranolol has been detected in United States wastewater effluents at concentrations ranging from 0.026 to 1.90 μg/l. In mammals, there is evidence that β-blockers can cause sexual dysfunction, and alter serotonergic pathways which may impact reproductive behavior but little is known about the effects on fish behavior. The present study tested the effects of propranolol on fecundity, on brain gene expression and on reproductive behavior of the fathead minnow, Pimephales promelas, a fish that exhibits male parental care. Sexually mature fathead minnows were housed at a ratio of one male and two females per tank and exposed to nominal concentrations of 0, 0.1, 1, 10 μg/l for 21 days. Measured concentrations (±SD) of propranolol were 0.003±0.004, 0.05±0.02, 0.88±0.34 and 4.11±1.19 μg/l. There were no statistically significant differences in fecundity, fertilization rate, hatchability and time to hatch. Propranolol exposure was not associated with a change in nest rubbing behavior, time spent in the nest or approaching the females. There was a significant difference in the number of visits to the nest with males receiving low and medium propranolol treatments. The microarray analysis showed that there were 335 genes up-regulated and 400 genes down-regulated in the brain after exposure to the highest dose of propranolol. Among those genes, myoglobin and calsequestrin transcripts (fold change=10.84 and 5.49, respectively) were highly up-regulated. Ontological analyses indicated changes in genes involved in calcium ion transport, transcription, proteolysis and apoptosis/anti-apoptosis. Pathway analysis indicated that the reduced expression of caspases may lead to impaired neurite outgrowth, neurotransmitter secretion and brain function in developing organisms. The results showed that exposure to propranolol at concentrations as high as 4.11 μg/l did not significantly impact reproductive behavior or spawning abilities of fathead minnow but did alter the regulation of genes within the brain of fish.
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Affiliation(s)
- Varenka Lorenzi
- Department of Environmental Sciences, University of California Riverside, Riverside, CA, USA
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Boncompagni S, Thomas M, Lopez JR, Allen PD, Yuan Q, Kranias EG, Franzini-Armstrong C, Perez CF. Triadin/Junctin double null mouse reveals a differential role for Triadin and Junctin in anchoring CASQ to the jSR and regulating Ca(2+) homeostasis. PLoS One 2012; 7:e39962. [PMID: 22768324 PMCID: PMC3388061 DOI: 10.1371/journal.pone.0039962] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2012] [Accepted: 05/31/2012] [Indexed: 11/17/2022] Open
Abstract
Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+) imaging and Ca(2+) selective microelectrodes we found that changes in e-c coupling, SR Ca(2+)content and resting [Ca(2+)] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+) regulation than Jct/CASQ association.
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Affiliation(s)
- Simona Boncompagni
- DNI-Department of Neuroscience and Imaging, CeSI-Center for Research on Ageing, University of G. D'Annunzio, Chieti, Italy.
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