Mechanism of myocardial damage induced by doxorubicin via calumenin-regulated mitochondrial dynamics and the calcium–Cx43 pathway

Abstract

BACKGROUND

The clinical application of doxorubicin (DOX) is limited by its potential to cause cardiac cardiotoxicity.

AIM

To investigate the correlation between calumenin (CALU) and mitochondrial kinetic-related proteins in rats with DOX cardiomyopathy.

METHODS

A rat model of DOX-induced cardiomyopathy was used to evaluate the effects of DOX. We observed the effect of DOX on electrical conduction in cardiomyocytes using the electromapping technique. Masson staining was performed to evaluate myocardium fibrosis. Electron microscopy was used to observe the changes in pathological ultrastructure of the myocardium. Western blotting and ELISAs were performed to detect protein levels and intracellular free Ca²⁺ concentration.

RESULTS

DOX slowed conduction and increased conduction dispersion in cardiomyocytes. The myocardial pathology in rats treated with DOX exhibited a significant deterioration, as demonstrated by an increase in mitochondrial Ca²⁺ concentration and a decrease in the expression of CALU, optic atrophy-1, and Bcl-2. Additionally, there was an increase in the expression of connexin 43 (Cx43) and the mitochondrial mitotic proteins dynamin-related protein 1, CHOP, Cytochrome C, and Bax in DOX rats. Decreased expression of CALU in cardiomyocytes triggered an increase in cytoplasmic free calcium concentration, which would normally be taken up by mitochondria, but decreased expression of mitochondrial outer membrane fusion proteins triggered a decrease in mitochondrial Ca²⁺ uptake, and the increase in cytoplasmic free calcium concentration triggered cell apoptosis.

CONCLUSION

Increased cytoplasmic free calcium ion concentration induces calcium overload in ventricular myocytes, leading to decreased Cx43 protein, slowed conduction in myocytes, and increased conduction dispersion, resulting in arrhythmias.

Key Words: Calumenin; Mitochondrial dynamics; Doxorubicin; Apoptosis; Ca²⁺ concentration; Cardiotoxicity

Core Tip: Doxorubicin (DOX) is an antitumor drug, with the main side effect being cardiotoxicity. This study investigated the time course and mechanism of DOX-induced myocardial injury by injecting DOX into rats and conducting cardiac electrophysiological tests and ultrastructural observations of myocardium. The results showed that DOX began to damage myocardial mitochondria as early as the second week after administration. The study clearly demonstrated that DOX causes calcium overload in myocardial cells by reducing calumenin expression, which in turn leads to myocardial cell apoptosis and arrhythmia.

INTRODUCTION

Doxorubicin (DOX), a widely used anthracycline antitumor agent, has been a cornerstone in the treatment of various malignancies, including breast cancer, lymphoma, and sarcoma, since its introduction in the 1960s. Despite its potent anticancer efficacy, the clinical application of DOX is substantially limited by its dose-dependent cardiotoxicity, which may result in irreversible cardiomyopathy and congestive heart failure. This condition, known as DOX-induced cardiomyopathy (AIC), remains a major concern in oncology, as it often necessitates the discontinuation of therapy or limits the cumulative dose that can be safely administered[1-3]. The pathogenesis of AIC is multifactorial and involves a complex interplay of molecular mechanisms. Early studies have highlighted the role of oxidative stress as a central driver of DOX-induced cardiotoxicity. Doxorubicin is known to undergo redox cycling, generating excessive reactive oxygen species (ROS) that overwhelm the endogenous antioxidant defenses of cardiomyocytes, leading to mitochondrial dysfunction, DNA damage, and lipid peroxidation[4]. More recently, emerging evidence has implicated additional mechanisms, including topoisomerase IIβ inhibition, which disrupts mitochondrial biogenesis and function, and activation of inflammatory pathways such as NF-κB and NLRP3 inflammasome signaling[5]. Dysregulation of autophagy, ferroptosis, and epigenetic modifications have also been identified as contributing factors to the progression of AIC[6]. Despite these advances, the precise molecular cascades underlying AIC remain incompletely understood, and effective strategies to prevent or mitigate doxorubicin-induced cardiotoxicity are still lacking.

Mitochondrial dynamics play an important role in the pathogenesis of various cardiomyopathies[7], and play an important role in maintaining the normal function of cardiomyocytes[8]. Due to the highly dynamic characteristics of mitochondria, they can continue the fusion–division cycle, which is known as mitochondrial dynamics. Mitochondrial fusion proteins include Mfn1 and Mfn2 and optic atrophy (OPA)-1, and mitochondrial division proteins include mitochondrial fission (Fis) 1 and dynamin-related protein (DRP)-1[9,10].

Connexin 43 (Cx43) is one of the most important gap junction proteins in cardiomyocytes, responsible for intercellular electrical signaling and material exchange, and essential for maintaining normal electrophysiological function and structural integrity of the heart[11]. It was found that in DOX-induced cardiomyopathy, Cx43 expression was decreased and abnormally distributed. Normally, Cx43 is mainly located in the intercalated disc region of cardiomyocytes, but after DOX treatment[12], it is closely associated with the development of myocardial fibrosis, which may lead to inhomogeneous electrical conduction and further increase the risk of arrhythmias[13]. Cx43 is closely associated with the development of myocardial fibrosis, which may lead to heterogeneity of electrical conduction, further increasing the risk of arrhythmias. DOX exacerbates cardiomyocyte injury by disrupting the function of mitochondrial Cx43, leading to mitochondrial ROS overproduction and impaired energy metabolism[13].

Calumenin (CALU), a member of the CREC family, is commonly found in the endoplasmic reticulum/sarcoplasmic reticulum of mammalian cardiomyocytes[14]. Decreased expression of CALU protein causes intracellular Ca²⁺ overload and leads to endoplasmic reticulum stress; however, CALU can alleviate the endoplasmic reticulum stress response of cardiomyocytes to a certain extent, and reduce the apoptosis rate of cardiomyocytes due to endoplasmic reticulum stress[15]. In recent years, the role of CALU in regulating calcium homeostasis in the endoplasmic reticulum has been widely recognized in studies of DOX[16-18]. With the increasing number of studies on DOX cardiomyopathy, these and other studies have suggested that its occurrence and development mechanism are complex. However, the etiology remains to be established by in vivo and in vitro experiments to explore the role of CALU in the pathogenesis of DOX cardiomyopathy, and to provide a theoretical basis for its treatment.

In this study, we observed the effects of DOX on electrical conduction in cardiomyocytes, as well as its effects on the expression of CALU[19,20], mitochondrial calcium concentration, mitochondrial fusion proteins and split proteins in cardiomyocytes using an electric mapping technique, to clarify whether DOX-induced arrhythmias are caused by reduction of CALU expression and overload of mitochondrial calcium, and to provide a new target for the treatment of DOX-induced arrhythmias.

MATERIALS AND METHODS

Materials

DOX was supplied by Solarbio Co. Ltd (Beijing, China). The antibodies of CALU, OPA-1, DRP-1, Bax, Bcl-2, CHOP, Cytochrome C, CX43, and GAPDH were purchased from Abcam Co. Ltd. An intracellular free Ca²⁺ concentration kit was obtained from Shanghai Haohai Biological Technology Co. Ltd. (Shanghai, China).

Animals and treatments

All rats were fed with a standard diet. The treatment of rats was in accordance with the NIH Guide for the Care and Use of Laboratory Animals and all protocols were approved by the Institutional Animal Care and Use Committee of Inner Mongolia University for Nationalities. Thirty-six clean grade male Sprague–Dawley rats aged 2–3 months and weighing 180–220 g (purchased from Changchun Yisi Experimental Animal Technology Co., China Ltd.) were randomly divided into the control (CON) group and DOX group at different time points (2, 4 and 8 weeks). The animals were fed at 20°C–25°C and 50%–65% relative humidity. The light and dark cycle was 12 hours, and there were no restrictions on diet. Before the experiment, the rats underwent 1 week of preconditioning. After 1 week of feeding, rats in the DOX groups were injected intraperitoneally with DOX (2 mg/kg) weekly. Rats in the CON group were gavaged with 0.9% NaCl weekly. This was done for 2, 4 and 8 consecutive weeks. The general state of the rats in the model group was observed, mainly the reaction of rats, diet, hair color, activity, excretion and body weight were recorded.

Isolated cardiac perfusion

Rats were weighed, injected intraperitoneally with sodium heparin (3125 U/kg), anesthetized with isoflurane for 15 min, and then killed. The rats were placed on the experimental table and the chest was opened in an inverted "T" shape to expose the heart. The lungs were lifted up with forceps and the heart was quickly cut off along the back of the lungs and placed in a glass Petri dish with precooled benchtop solution[21]. The aorta was quickly located, excess tissue was cut away, the aorta was carefully placed at the bottom of the cannula, tied tightly with surgical sutures, and the pre-prepared KH fluid in the syringe was gently pushed into the heart to pump out the cardiac remnants for Langendorff perfusion. The perfusion rate was 10 mL/min and the perfusion temperature was 37°C ± 0.5°C. The residual blood in the heart was drained to restore the heart to normal rhythm with a heart rate > 250 bpm and stabilized for 15 minutes before the experiments[22].

Electrocardiographic labeling

The electrocardiogram (ECG) electrodes were placed on both sides of the heart and the ECG was recorded continuously; the pen test electrode was placed in the left ventricle and electrical signal conduction in the ventricular myocytes of rats in the model group was detected under sinusoidal conditions and compared with that of the CON group.

Masson staining

Rat left ventricular tissue was resected, fixed with paraformaldehyde solution, and Masson staining was performed. Changes in the degree of myocardial fibrosis of rats in each group were observed by microscopy (Nikon Eclipse CI).

Electron microscopy

Rat left ventricular tissues were excised and fixed in 4% glutaraldehyde solution in 2%–4.1% osmic acid and 0.1M PBS (pH 7.4) and fixed at 20°C for 2 hours. The tissue samples underwent dehydration, penetration, embedding, slicing and uranium lead double staining. The results were observed by electron microscopy (Tecnai G2 20 Twin).

Western blotting assay

Proteins were extracted from rat cardiomyocytes and newborn rat cardiomyocytes. After SDS-PAGE separation, the proteins were sealed with 5% BSA, and the primary anti-CALU (1:1000), anti-CHOP (1:1000), anti-DRP-1 (1:1000), anti-OPA-1 (1:1000), anti-CytC (1:1000), anti-Bax (1:1000), anti-Cx43 (1:1000) and anti-Bcl-2 (1:1000) antibodies were added and incubated at 4°C overnight. The secondary antibody was added and incubated for 1 h. Bound antibody was observed using enhanced chemiluminescence.

Determination of intracellular free Ca²⁺

The intracellular free Ca²⁺ in rat cardiac tissues was detected with the intracellular free Ca²⁺ detection kit and fluorescence labeling instrument, which were obtained from Shanghai Haohai Biological Technology Co. Ltd. Cardiac tissues were digested to a single-cell suspension, which was treated with reaction reagent and a dye solution. The results were detected with a fluorescence microplate instrument (excitation = 550 nm, emission = 590 nm).

Statistical analysis

The experimental data were expressed as mean ± SD. Statistical analysis was conducted using SSPS version 11.5 software. The t test was used to detect significant differences between the two groups. Student's unpaired or paired t test was performed for comparisons between two groups and multiple group comparisons were carried out using one-way ANOVA with Bonferroni post hoc test.

RESULTS

DOX decreased weight but increased heart–body weight ratio in rats

In the CON group, rats showed an increase in body weight over time, smooth and shiny fur and no mortality. However, the body weight of rats in the DOX-treated groups (2-week, 4-week, and 8-week groups) decreased significantly over time compared with the CON group (Figure 1A). The heart–body weight ratio was also significantly increased in the DOX-treated group, and this further increased with the duration of treatment (Figure 1B). These results suggest that DOX not only causes weight loss, but may also cause cardiac hypertrophy or edema.

Figure 1 Doxorubicin induced changes. A: Doxorubicin induced changes in body weight; B: Doxorubicin induced changes in heart weight of rats in each group; C: Doxorubicin caused pathological damage in the myocardium; D: Doxorubicin caused changes in collagen fibrosis rate. The results of Masson staining were observed under a microscope (40 ×) (n = 6). Data are expressed as mean ± SD, ^bP < 0.01, ^cP < 0.001 vs control group (n = 6). DOX: Doxorubicin; CON: Control.

DOX causes pathological damage in the myocardium

Pathological analysis of myocardial tissue showed that cardiomyocytes in control rats were tightly arranged and orderly, with fewer intercellular collagen fibers, and no significant fibrosis was observed. However, in the DOX -treated group, cardiomyocytes were disorganized and significantly deformed, with increased cell spacing, a significant increase in intercellular blue collagen fibers, and worsening fibrosis. In particular, myocardial fibrosis was most severe in the 8-week DOX-treated group (Figure 1C). These results confirmed the direct damaging effect of DOX on myocardial tissue.

DOX prolonged ventricular conduction time and conduction dispersion

The left ventricular signals of rat hearts were recorded with 64-channel pen electrodes. Under sinus rhythm, the effect of ventricular conduction time was obvious in the model group (Figure 2A), and the conduction changes were significantly different. Analysis of the left ventricular conduction time revealed that there was a tendency to prolong the conduction time after drug administration (Figure 2B), and there was no significant change in conduction velocity (Figure 2C).

Figure 2 Effects of doxorubicin on cardiac conduction and conduction dispersion. A: Representative plots of prolonged ventricular conduction time; B: Plot of changes in left ventricular conduction time in sinus conditions; C: Representative graphs of changes in left ventricular conduction velocity; D: Comparison of conduction dispersion maps; E: Comparison of conductance discrete bar charts. DOX: Doxorubicin; CON: Control; LV: Left ventricular.

Conduction dispersion was a concern in the study of arrhythmic disease mechanisms, and examination of cardiac conduction dispersion in DOX model rats in sinus rhythm revealed a significant effect on cardiac conduction dispersion in the model group (Figure 2D), with a tendency for prolongation of ventricular conduction dispersion in sinus rhythm (Figure 2E).

Effect of DOX on cardiac ECG in rats

ECG is an important component of cardiac monitoring, and cardiac ECG changes were determined using multichannel electrophysiological labeling assays (Figure 3A). Heart rate slowed down after drug administration and all changes were significantly different (Figure 3B). Statistical analysis revealed prolongation of the QT interval (Figure 3C). When the QT interval was prolonged, the action potential timescale was abnormally prolonged, which may have led to increased Ca²⁺ inflow via L-type Ca²⁺ channels. Dysfunction of IK1 channels may have affected the function of other ion channels (e.g., IKr), which indirectly led to prolongation of the QT interval.

Figure 3 Effect of doxorubicin on the electrocardiogram of rat heart and the expression of connexin 43. A: Representative graph of left ventricular field potentials; B: Heart rate variability; C: QT variations. DOX: Doxorubicin.

DOX increased the concentration of mitochondrial Ca²⁺ and enhanced expression of Cx43

Mitochondrial Ca2+ levels rose significantly in the DOX group compared to the CON group, both in vivo and in vitro. Levels were also higher in the 8-week DOX group than in the 2-week and 4-week DOX groups (Tables 1 and 2).

Table 1 The concentration of intracellular free Ca²⁺ of cardiomyocytes in vivo.

Group	The concentration of intracellular free Ca2+ (nmol/L)
CON	18.62 ± 3.21 (nmol/L)
2 weeks DOX	56.45 ± 11.3 (nmol/L)b
4 weeks DOX	87.61 ± 9.65 (nmol/L)c
8 weeks DOX	101.21 ± 10.14 (nmol/L)c

^cP < 0.001.

^bP < 0.01 vs CON (n = 6).

mean ± SD was analyzed. DOX: Doxorubicin; CON: Control.

Table 2 The concentration of intracellular free Ca²⁺ of cardiomyocytes in vitro.

Group	The concentration of intracellular free Ca2+ (nmol/L)
CON	20.14 ± 1.31 (nmol/L)
DOX	90.89 ± 23.15 (nmol/L)c

^cP < 0.001.

mean ± SD was analyzed. DOX: Doxorubicin; CON: Control.

Western blotting revealed that the levels of Cx43 protein were significantly elevated in the DOX group compared with the CON group (Figure 4).

Figure 4 Effect of doxorubicin on expression of connexin 43. Levels of Cx43 were measured by western blotting. Data are expressed as mean ± SD, ^cP < 0.001 vs control group (n = 6) and (n = 3). DOX: Doxorubicin; CON: Control.

DOX affected the ultrastructural of myocardial tissue

In the intact sarcomere, it was observed through the use of electron microscopy that the heart tissue of the CON rat exhibited a well-preserved structure, featuring clearly visible mitochondria and myocardial fibers that were neatly arranged in an orderly fashion. Ultrastructural changes in cardiomyocytes were observed in DOX-treated rats. Compared with the CON group, disordered myocardial fibers and vacuolation of mitochondria were found in the DOX groups. Vacuoles in mitochondria disappeared and myocardial fibers were fractured, few and even absent in the 8-week DOX group, compared with the 2-week and 4-week DOX groups (Figure 5).

Figure 5 Doxorubicin altered the ultrastructural of myocardial tissue. Transmission electron microscopy results, and cell morphology under inverted microscope (1.0 μm) (n = 6). A: Control; B: Doxorubicin (DOX) (2w); C: DOX (4w); D: DOX (8w).

DOX mediated myocardial injury by influencing mitochondrial dynamics

DOX reduced the expression of CALU in vivo in the DOX group, compared with the CON group (Figure 5). OPA-1 was decreased and DRP-1 was increased in the DOX group, compared with the CON group (Figure 6). To confirm the role of mitochondrial-induced apoptosis, we analyzed the expression of CytC, CHOP, Bax and Bcl-2. DOX increased expression of CytC, CHOP and Bax and reduced expression of Bcl-2 in the DOX group (Figure 7).

Figure 6 Doxorubicin influenced mitochondrial dynamics related proteins. A: Protein levels of calumenin, optic atrophy 1 and dynamin-related protein 1 were measured by western blotting; B: Protein levels of calumenin; C: Protein levels of optic atrophy 1; D: Protein levels of dynamin-related protein 1. Data are expressed as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control (n = 6) and (n = 3). DOX: Doxorubicin; CON: Control; OPA-1: Optic atrophy 1; DRP-1: Dynamin-related protein 1.

Figure 7 Doxorubicin attenuated myocardial injury by inhibiting mitochondrial induced apoptosis. A: Protein levels of CHOP, cytochrome C, Bax and Bcl-2 were measured by western blotting; B: Protein levels of CHOP; C: Protein levels of cytochrome C; D: Protein levels of bax; E: Protein levels of Bcl-2. Data are expressed as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group (n = 6) and (n = 3). CytC: Cytochrome C; DOX: Doxorubicin; CON: Control.

DISCUSSION

The current study demonstrates that in rat hearts, the effect of DOX in vivo may be associated with CALU-regulated mitochondrial dynamics and the calcium-Cx43 pathway. Related studies[20,23,24] have shown that DOX can cause pathological damage in the myocardium. In our study, we found that cardiomyocyte fibrosis was more severe in the DOX group than in the CON group, and mitochondrial vacuoles disappeared and myocardial fibers were fractured and fewer in rat hearts treated with DOX, compared with control rat hearts. These findings suggested that the effect of DOX on the heart was related to mitochondria. Mitochondrial dynamics play a crucial role in physiological functions, including the maintenance of mitochondrial DNA stability and critical life processes like energy synthesis and cell aging[19]. Mitochondrial function is recognized as an effective treatment target for heart failure, as agreed upon by experts[25]. Studies have shown that Ca²⁺ overload in cells can induce increased expression of DRP-1[21,26], downregulate expression of mitochondrial fusion proteins Mfn1 and Mfn2, and produce large amounts of CytC and activate apoptosis pathways such as caspase-3, which leads to increased apoptosis[27,28]. Thus, understanding the role of mitochondrial-fusion-division imbalance in heart failure is crucial for its treatment. In our study, we found that intracellular Ca²⁺ concentration in the DOX group was significantly higher than that in the CON group. This suggested that DOX causes intracellular Ca²⁺ overload.

Calcium plays a necessary role in nerve impulse transmission, cell signaling, and maintaining calcium homeostasis in mammals. In our research, we discovered that DOX prolonged ventricular conduction time and conduction dispersion, and slowed heart rate. In addition, statistical analyses revealed that DOX prolonged the QT interval. The key structure for rapid electrical conduction between cardiomyocytes is the gap junction, a transmembrane channel for electrical and chemical coupling between cardiomyocytes, which is essential for maintaining cardiac rhythm, of which Cx-forming hemichannels are a major component. Cardiomyocytes express Cx40, Cx43 and Cx45, with Cx43 being the most widely distributed in the mammalian cardiovascular and nervous systems, and the main Cx expressed by ventricular myocytes. The mechanism by which DOX leads to the reduction of Cx43 protein involves several aspects, including oxidative stress, abnormal phosphorylation regulation, and activation of protein degradation pathways. DOX undergoes redox cycling in cells through its quinone structure, generating large amounts of ROS[29]. Excess ROS directly damage cardiomyocytes, and may also lead to structural and functional impairment of Cx43 by oxidizing key amino acid residues (e.g., cysteine and methionine) of the Cx43 protein[29]. DOX inhibits the activity of protein kinase (PK) A and PKC, which normally stabilize Cx43 by phosphorylating specific serine residues (e.g., Ser368 and Ser365) to stabilize Cx43 and promote its localization to the cell membrane[30]. In contrast, DOX may activate protein phosphatases (e.g., PP1 and PP2A), leading to dephosphorylation of Cx43, which in turn promotes its degradation via the ubiquitin–proteasome pathway[31]. It has been shown that DOX can upregulate the expression of E3 ubiquitin ligases (e.g. Nedd4 and ITCH) that recognize and ubiquitinate Cx43, thereby promoting its degradation by the proteasome[32]. DOX may also induce autophagic degradation of Cx43 by activating autophagy-related proteins such as LC3 and p62[33]. Mitochondria are the main source of intracellular ROS, and DOX leads to overproduction of ROS by disrupting the mitochondrial electron transport chain, which in turn affects the stability of Cx43[34]. In this study, western blotting showed that DOX enhanced expression of Cx43.

It has been reported[22] that CALU plays an important role in the occurrence and development of acute myocardial ischemia, arrhythmia and other diseases. Our results also showed that expression of CALU was significantly decreased in the DOX group compared with the CON group. This indicated that expression of CALU was decreased by DOX. We found that DOX decreased rat body weight, and increased the heart weight–body weight ratio. DOX reduced expression of CALU due to Ca²⁺ overload, and increased apoptosis of myocardial cells. In vivo experiments showed that DOX reduced expression of CALU, OPA-1 and Bcl-2, and increased expression of DRP-1, CytC, CHOP and Bax in rats.

CONCLUSION

We found that DOX decreased expression of CALU and mitochondrial fusion proteins, increased expression of mitochondrial split proteins, and increased the cytoplasmic calcium concentration of cardiomyocytes. CALU is a calcium-binding protein found in the lumen of the endoplasmic reticulum. If the expression of CALU is reduced in cardiomyocytes, calcium ions from the endoplasmic reticulum leak into the cytoplasm, which leads to an increase in the cytoplasmic concentration of free calcium ions. Under normal circumstances, the increased concentration of free calcium ions in the cytoplasm is absorbed by the mitochondria, but the decreased expression of mitochondrial outer membrane fusion proteins leads to a decrease in Ca²⁺ uptake by the mitochondria, and the increased concentration of free calcium ions in the cytoplasm triggers apoptosis. At the same time, the increase in cytoplasmic free calcium ion concentration induces calcium overload in ventricular myocytes, which leads to a decrease in Cx43 protein, a slowing of conduction in myocytes, and an increase in conduction dispersion, resulting in arrhythmias. Decreased expression of CALU, an endoplasmic reticulum-resident calcium-binding protein, may be associated with DOX-induced oxidative stress, endoplasmic reticulum stress, and aberrant transcriptional regulation. First, DOX causes oxidative stress by generating large amounts of ROS, which may directly impair mRNA or protein stability in CALU. Second, DOX may inhibit transcription or translation of CALUs by activating endoplasmic reticulum stress responses such as the PERK/ATF4 and IRE1/XBP1 pathways. In addition, DOX may indirectly downregulate CALU expression by affecting the activity of transcription factors.

Future studies will include female rats and systematically compare the differences in DOX cardiotoxicity in male and female rats. This may involve the role of sex hormones (e.g., estrogen and androgen) in the regulation of CALU and Cx43 expression, as well as the effect of sex on mitochondrial dynamics and calcium homeostasis. We plan to design experiments with multiple dose groups (e.g., low, medium and high doses) and multiple time points (e.g., short-term and long-term) in future studies to assess the dose–response relationship and cumulative effects of DOX. This will contribute to a more comprehensive understanding of the dose-dependent and time-dependent characteristics of DOX cardiotoxicity. We plan to explore the following interventions in follow-up studies: CALU-based therapies: Restoration of CALU expression and function by gene overexpression or pharmacological interventions (e.g., calcium homeostasis modulators). Modulation of mitochondrial dynamics: Improving mitochondrial function using mitochondrial fusion promoters (e.g., M1) or fission inhibitors (e.g., Mdivi-1). Antioxidant and anti-inflammatory strategies: Attenuate DOX-induced oxidative stress and inflammatory responses using antioxidants (e.g., N-acetylcysteine) or anti-inflammatory agents (e.g., IL-1 receptor antagonists).