To summarize and critically assess the contribution of genetics to the Long QT Syndrome (LQTS), with specific reference to the unraveling of its underlying mechanisms and to its impact on clinical practice.

The evolution towards our current approach to therapy for LQTS patients is examined in terms of risk stratification, gene-specific management, and assessment of the clinical impact that genetic modifiers may have in modulating the natural history of the patients. Glimpses are provided on the newest multidisciplinary approaches to study disease mechanisms, test new candidate drugs and identify precision treatments.

It is undeniable that genetics has revolutionized our mechanistic understanding of cardiac channelopathies. Its impact has been enormous but, curiously, the way LQTS patients are being treated today is largely the same that was used in the pregenetic era, even though management has been refined and gene-specific differences allow a more individually tailored antiarrhythmic protection. The synergy of genetic findings with modern in vitro and in silico tools may expand precision treatments; however, they will need to prove more effective than the current therapeutic approaches and equally safe.

The existence of an important relationship between stress, the autonomic nervous system, and sudden cardiac death (SCD) has been long recognized. In the present essay we review the large number of conditions, acting at individual or at population level, that have been causally associated to SCD and discuss the mechanistic and translational value of the studies exploring such associations. These conditions include external stressors (earthquakes, wars) and internal stressors (anger, fear, loss of a loved one) and emotions of even opposite sign. Most situations confirm the time-honored view that increases in sympathetic activity are proarrhythmic whereas increases in vagal activity are protective; however, we will also show and discuss a condition in which the culprit appears to be the excess of vagal activity. The physiologic rationale underlying the most typical situations is on one hand the profibrillatory effect of the increase in the heterogeneity of repolarization secondary to the release of norepinephrine, and on the other the combined effect of acetylcholine to lower heart rate and to antagonize the cardiac effects of norepinephrine at ventricular level. An interesting facet of this potentially lethal relationship is that the elements involved are by no means always exceptional, and they can actually represent part of our everyday life.

Mitochondrial cardiomyopathy (MCM) is characterized as an oxidative phosphorylation disorder of the heart. More than 100 genetic variants in nuclear or mitochondrial DNA have been associated with MCM. However, the underlying molecular mechanisms linking genetic variants to MCM are not fully understood due to the lack of appropriate cellular and animal models. Patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) provide an attractive experimental platform for modeling cardiovascular diseases and predicting drug efficacy to such diseases. Here we introduce the pathological and therapeutic studies of MCM using iPSC-CMs and discuss the questions and latest strategies for research using iPSC-CMs.

Ethical concerns about stem cell-based research have delayed important advances in many areas of medicine, including cardiology. The introduction of induced pluripotent stem cells (iPSCs) has supplanted the need to use human stem cells for most purposes, thus eliminating all ethical controversies. Since then, many new avenues have been opened in cardiology research, not only in approaches to tissue replacement but also in the design and testing of antiarrhythmic drugs. This methodology has advanced to the point where induced human cardiomyocyte cell lines can now also be obtained from commercial sources or tissue banks. Initial studies with readily available iPSCs have generally confirmed that their behavioral characteristics accurately predict the behavior of beating cardiomyocytes in vivo. As a result, iPSCs can provide new ways to study arrhythmias and heart disease in general, accelerating the development of new, more effective antiarrhythmic drugs, clinical diagnoses, and personalized medical care. The focus on producing cardiomyocytes that can be used to replace damaged heart tissue has somewhat diverted interest in a host of other applications. This manuscript is intended to provide non-specialists with a brief introduction and overview of the research carried out in the field of heart rhythm disorders.

Recent clinical studies that evaluated the effects of supplemental omega-3 polyunsaturated fatty acids (n-3 PUFAs) on sudden cardiac death have yielded conflicting results. Our aim was to clarify this issue using an established and clinical relevant canine model of sudden cardiac death.

Susceptibility to ventricular fibrillation (VF) was evaluated using a 2-minute left circumflex artery occlusion during the last minute of an exercise test in 76 dogs (from 2 independent studies) with healed myocardial infarctions (MI); 44 developed VF (susceptible, VF+), whereas 32 did not (resistant, VF-). These dogs were then randomly assigned to either placebo (1 g/d, corn oil; 15 VF+, 11 VF-) or n-3 PUFA (1-4 g/d, docosahexaenoic acid+eicosapentaenoic acid ethyl esters, 29 VF+, 21 VF-) groups. Seven sham (no-MI) dogs were also treated with n-3 PUFA (4 g/d). After treatment (3 months), the exercise+ischemia test was repeated. Dietary n-3 PUFAs produced significant (P<0.01) increases in red blood cell and left ventricular n-3 PUFA levels. Nine post-MI (5 placebo versus 4 n-3 PUFA) and 2 sham dogs died suddenly during the 3-month treatment period. The n-3 PUFA treatment failed to prevent arrhythmias in VF+ dogs (decreased in 27% placebo versus 24% n-3 PUFA, P=0.5646) but induced VT/VF in VF- animals (n-3 PUFA 33% versus placebo 0%, P=0.0442).

Despite large increases in cardiac tissue n-3 PUFA content, dietary n-3 PUFAs did not prevent ischemia-induced VF and actually increased arrhythmia susceptibility in both noninfarcted and low-risk post-MI dogs.

Amiodarone is one of the most effective antiarrhythmic drugs. However, poor solubility of this compound has limited its intravenous application. SAR11464A is a water-soluble amiodarone-like drug that lacks iodine and inhibits multiple cardiac ion channels in vitro. This study evaluated the antiarrhythmic efficacy of this drug in vivo. In porcine studies, atrial effective refractory period (AERP) was measured in pentobarbital-anesthetized thoracotomized pigs and atrial fibrillation (AF) was induced by a premature beat. Ventricular fibrillation (VF) was induced via either burst pacing or programmed electrical stimulation (a series of progressively shorter beats, S1-S5). In canine studies, VF was induced by a 2-min occlusion of the left circumflex coronary artery during the last minute of exercise in dogs with healed myocardial infarctions (n = 8). One week later, this test was repeated after pretreatment with SAR114646A (3.0 mg/kg, i.v., slow bolus). SAR114646A produced a significant dose-dependent prolongation of AERP, inhibited AF induced by a premature stimulus, and electrically induced VF in anesthetized pigs. At 1.0 and 3.0 mg/kg, i.v., it was superior to amiodarone, dofetilide, and flecainide. In dogs, SAR114646A did not alter any ECG parameter including QTc (control, 236.9 ± 8.5 ms vs. SAR, 237.2 ± 3.5 ms) but significantly reduced the incidence of VF, protecting six of eight animals (Fisher's exact test, P = 0.01). SAR114646A was effective against both atrial and ventricular arrhythmias without altering ventricular repolarization. These data suggest that the amiodarone-like drug SAR114646A may be an effective antiarrhythmic intervention that does not adversely prolong ventricular repolarization.

Damaged heart muscle has only a minimal ability for regeneration following myocardial infarction in which cardiomyocytes are lost to ischemia. The most clinically promising approach to regeneration of cardiac muscle currently under investigation is that of injecting cardiogenic repair cells or implanting a preformed tissue-engineered patch. While major advances are being made in the derivation of functional human cardiomyocytes and the development of tissue-engineering modalities for cardiac repair, the host environment into which the repair cells are placed is largely overlooked. Within seconds of myocardial ischemia, hypoxia sets in in the myocardium and the inflammatory response starts, characterized by rapid deployment of circulating cells and the release of paracrine and autocrine signals. Therefore, the inflammatory conditions under which these interactions take place, the design of the scaffold material used, and the maturity of the implanted cells will determine the outcomes of any stem cell-based therapy. We discuss here the interactions between implanted and inflammatory cells of the host, which are critical for the design of effective heart repair therapies.

Unexpected sudden death among apparently healthy individuals remains a daunting problem. We have previously shown that autonomic modulation of cardiac arrhythmias and autonomic markers, such as baroreflex sensitivity (BRS) and heart rate variability (HRV), carry predictive power after myocardial infarction.

We tested the hypothesis that a parameter combining BRS and HRV could predict risk for ventricular fibrillation (VF) during a first ischemic episode in otherwise healthy dogs.

In 43 fully instrumented dogs, BRS and frequency domain analysis of HRV were determined, as well as the occurrence (n = 10, high-risk) or absence (n = 33, low-risk) of VF during 2 minutes of myocardial ischemia superimposed on submaximal exercise. TARVA (Tonic and Reflex Vagal Activity), expressed in units, is the parameter resulting from the multiplication of BRS by HF/LF (an index of tonic vagal activity).

High-risk dogs had markedly lower TARVA values, reflecting lower cardiac vagal activity, than low-risk animals (12 +/- 5 versus 56 +/- 43 units, P < .001). The area under the receiver-operator characteristic curve for TARVA was 0.96 (95% confidence interval 0.86 to 0.99); its optimal cutoff had a 100% sensitivity and a 88% specificity with positive and negative predictive values of 71% and 100%, respectively.

Differences in cardiac autonomic activity, present in healthy dogs, allow prediction of arrhythmic risk during a first ischemic episode. Increased risk is associated with reduced vagal activity. If confirmed in humans, this finding would open the way to the identification of those apparently healthy subjects at risk for sudden cardiac death during their first episode of myocardial ischemia.

The purpose of this study was to examine the sympathetic-parasympathetic interactions on heart rate through release of neuropeptide Y (NPY) and its action on prejunctional NPY Y2 receptors on vagal and sympathetic nerve fibers. In other studies on various preparations and in various organs, attenuation of transmitter release has in fact been demonstrated through activation of the NPY Y2 receptor. In the present study on anesthetized dogs we examine, however, for the first time if vagal bradycardia is attenuated by endogenous NPY released during intense cardiac sympathetic stimulation. In addition, we explore if sympathetic transmitter release and heart rate, during moderate sympathetic stimulation, are affected through this receptor system. The significance of the NPY Y2 receptor was revealed by performing experiments before and after administration of its specific receptor antagonist BIIE0246. We found that attenuation of the bradycardia during vagal nerve stimulation was dose-dependently counteracted by BIIE0246 and that the tachycardia elicited by sympathetic stimulation remained unaffected after NPY Y2 receptor blockade. Thus, endogenous NPY appears to attenuate vagal bradycardia by stimulating prejunctional NPY Y2 receptors on cardiac vagal nerve terminals and, less efficiently, to attenuate transmitter release and tachycardia through a feedback loop on the cardiac sympathetic nerve fibers.

This study examined the hypothesis that the sympathomimetic activity of ephedrine increases the risk of lethal arrhythmias.

The sympathomimetic amine, ephedrine, is used to augment physical performance and as a weight loss aid, but little is known about the cardiovascular consequences in individuals with ischemic heart disease.

Fifteen dogs at low risk for ventricular fibrillation (VF) during exercise and transient myocardial ischemia 30 days after a small anterior myocardial infarction were retested after five days of ephedrine use (Xenadrine, 0.4 mg/kg/day orally). To assess the effects of ephedrine on cardiac autonomic control, baroreceptor reflex sensitivity (BRS), heart rate (HR) variability, HR response to acute myocardial ischemia, and resting catecholamines were measured before and after ephedrine. Dogs were used as their own control when possible.

Nine of 15 animals had increased ventricular arrhythmias during ephedrine treatment (p = 0.01) and four had VF. Three dogs that had VF could not be resuscitated. Five animals with increased arrhythmias during ephedrine treatment had none during a third exercise and ischemia test after drug washout. Heart rates were higher after 30 s of myocardial ischemia during ephedrine treatment (204 +/- 25 beats/min no drug vs. 218 +/- 26 beats/min with ephedrine, p = 0.03). All plasma catecholamines increased after ephedrine administration. No changes in BRS, HR variability, or exercise HR were noted.

Ephedrine increases ischemia-dependent arrhythmias at doses recommended in over-the-counter preparations. Increased arrhythmia risk was associated with augmented ischemia-dependent sympathetic reflex activation.

Chronic Chagas' disease cardiomyopathy (CCC) is caused by the protozoan Trypanosoma cruzi, and it affects 30% of the 16-18 million people infected in Latin America. A good rodent model that develops a dilated cardiomyopathy closely resembling human CCC after T. cruzi infection is still needed. We compared the cardiomyopathy developed by T. cruzi-infected Syrian hamsters with human Chagas' disease cardiomyopathy using quantitative methods. Female hamsters were infected with 3.5 x 10(4) (G1, n = 10) or 10(5) (G2, n = 10) T. cruzi Y strain blood trypomastigotes. Control animals (C, n = 10) were injected with saline solution. Cardiac function was assessed by echocardiography at 4, 8 and 12 months post-infection. Heart sections were submitted to histopathological/morphometric analysis 12 months post-infection. At this time, ventricular dysfunction and diffuse or multi-focal myocarditis were observed in 91% and 100% of G1 and G2 infected groups, respectively. Median interstitial collagen volumes in groups C, G1 and G2 were 1.2%, 1.9% and 3.9%, respectively, and were significantly higher in group G2 than in group C. Among infected animals, myocarditis showed a positive correlation with interstitial fibrosis. Deaths in the chronic phase (8-12 months post-infection) were more frequent among G2 than G1, and were associated with macroscopic ventricular dilation, severe myocarditis and increased fibrosis values, along with an earlier onset of ventricular dysfunction. The T. cruzi chronically infected Syrian hamster develops a cardiomyopathy which resembles human Chagas' disease cardiomyopathy, and might be an adequate tool to investigate pathogenic mechanisms of this disease and to search for novel therapeutic strategies.

Anti-arrhythmic compounds with multiple actions reduce arrhythmic death risk in post-myocardial infarction (MI) patients. Sudden death prevention, however, may rely more on implantable defibrillators than anti-arrhythmic drugs due to ineffective pharmacologic intervention. Widespread use of implantable defibrillators should not obscure the need for development of new anti-arrhythmic drugs. This study tested the hypothesis that combined blockade of I(Na) and I(Ca(L)) prevents ischemia-dependent ventricular fibrillation (VF) in conscious dogs after MI. I(Na) and I(Ca(L)) blockade was accomplished with levosemotiadil in 11 dogs known to be at high risk for VF during 2 min of coronary occlusion during submaximal treadmill exercise 30 days after MI. Negative chronotropic effect of levosemotiadil was examined using the heart rate response to isoproterenol and comparing it with response to propranolol. Levosemotiadil prevented VF in 64% (7 of 11) of the high-risk animals. Heart rate responses to myocardial ischemia and to graded doses of isoproterenol were blunted by the high dose of levosemotiadil. Propranolol prevented VF in 73% (8 of 11) of the dogs. Levosemotiadil had approximately one half the beta-blocking activity of propranolol. The combination of I(Na) and I(Ca(L)) channel blockade coupled with partial beta-adrenergic blockade was equally effective in preventing VF as propranolol.

Suppression of malignant ventricular arrhythmias by selective blockade of the cardiac slowly activating delayed rectifier current (I(Ks)) has been demonstrated with the benzodiazepine L-768673 [(R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide] in canine models of recent and healed myocardial infarction. The present study extends the initial antiarrhythmic assessment of I(Ks) blockade by demonstrating prevention of ischemic malignant arrhythmias in dogs with recent (8.0 +/- 0.4 days) anterior myocardial infarction with the coadministration of a subeffective dose of L-768673 and a subeffective, minimally beta-adrenergic blocking dose of timolol. Administered individually, neither 0.3 microg/kg i.v. L-768673 nor 1.0 microg/kg i.v. timolol prevented the induction of ventricular tachyarrhythmia (VT) by programmed ventricular stimulation (PVS) or the development of malignant ventricular arrhythmia in response to acute coronary artery thrombosis. In contrast, coadministration of 0.3 microg/kg i.v. L-768673 + 1.0 microg/kg i.v. timolol suppressed the induction of VT by PVS (8/10, 80% rendered noninducible versus 1/10, 10% noninducible in vehicle group; p < 0.01) and prevented the development of acute ischemic lethal arrhythmias (3/10, 30% incidence versus 8/10, 80% incidence in vehicle group; p < 0.05). Concomitant administration of low-dose L-768673 + timolol produced modest increases in QTc and paced QT intervals (4.5 +/- 1.2 and 5.5 +/- 1.4%; both p < 0.01), increases in noninfarct zone relative and effective refractory periods (7.0 +/- 1.7 and 12.3 +/- 3.0%; both p < 0.01), and lesser increases in infarct zone relative and effective refractory periods (5.3 +/- 1.6 and 5.8 +/- 1.4%; both p < 0.01). These findings suggest that concomitant low-dose I(Ks) and beta-adrenergic blockade may constitute a potential pharmacologic strategy for prevention of malignant ischemic ventricular arrhythmias.

Using a model of arrhythmias associated with ischemic left ventricular (LV) dysfunction, this study investigated autonomic and electrophysiologic mechanisms associated with sudden cardiac death (SCD) in chronic heart failure (HF).

Left ventricular dysfunction from ischemic heart disease is associated with many instances of SCD. Electrophysiologic and autonomic derangements occur in HF, but their contribution to SCD risk is poorly understood.

Sudden death risk was assessed in 15 dogs with a healed anterior myocardial infarction (MI) during submaximal exercise and brief acute circumflex ischemia. Left ventricular dysfunction was then induced by repetitive circumflex microembolization until LV ejection fraction reached 35%. Before embolization, six dogs were susceptible to SCD, and nine were resistant.

Baroreflex sensitivity was lower in susceptible dogs (10 ms/mm Hg +/- 4 ms/mm Hg vs. 20 ms/mm Hg +/- 11 ms/mm Hg, p = 0.04). QT intervals from susceptible dogs were longer after MI (246 ms +/- 26 ms susceptible vs. 231 ms +/- 20 ms resistant, p < 0.001) and prolonged within eight weeks after LV dysfunction was established (from 246 ms +/- 26 ms to 274 ms +/- 56 ms, +11%, p < 0.01). Heart rate in susceptible dogs increased during transient ischemia (+20%) and with progressive LV dysfunction (102 beats/min +/- 28 beats/min baseline to 154 beats/min +/- 7 beats/min LV dysfunction, p = 0.003). All susceptible dogs had spontaneous sustained ventricular tachycardia culminating in SCD. In contrast, QT intervals in resistant dogs prolonged after 24 +/- 6 weeks (from 231 ms +/- 20 ms to 238 ms +/- 15 ms, p < 0.05), and heart rates were unchanged. Only one resistant dog died suddenly with LV dysfunction.

Depressed vagal and elevated sympathetic control of heart rate coupled with abnormal repolarization are associated with high SCD risk when post-MI LV dysfunction develops.

Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.

The factors that contribute to the occurrence of sudden cardiac death (SCD) in patients with chronic myocardial infarction (MI) are not entirely clear. The present study tests the hypothesis that augmented sympathetic nerve regeneration (nerve sprouting) increases the probability of ventricular tachycardia (VT), ventricular fibrillation (VF), and SCD in chronic MI. In dogs with MI and complete atrioventricular (AV) block, we induced cardiac sympathetic nerve sprouting by infusing nerve growth factor (NGF) to the left stellate ganglion (experimental group, n=9). Another 6 dogs with MI and complete AV block but without NGF infusion served as controls (n=6). Immunocytochemical staining revealed a greater magnitude of sympathetic nerve sprouting in the experimental group than in the control group. After MI, all dogs showed spontaneous VT that persisted for 5.8+/-2.0 days (phase 1 VT). Spontaneous VT reappeared 13.1+/-6.0 days after surgery (phase 2 VT). The frequency of phase 2 VT was 10-fold higher in the experimental group (2.0+/-2.0/d) than in the control group (0.2+/-0.2/d, P<0.05). Four dogs in the experimental group but none in the control group died suddenly of spontaneous VF. We conclude that MI results in sympathetic nerve sprouting. NGF infusion to the left stellate ganglion in dogs with chronic MI and AV block augments sympathetic nerve sprouting and creates a high-yield model of spontaneous VT, VF, and SCD. The magnitude of sympathetic nerve sprouting may be an important determinant of SCD in chronic MI.

To date, the lack of potent and selective inhibitors has hampered the physiological assessment of modulation of the cardiac slowly activating delayed rectifier current, I(Ks). The present study, using the I(Ks) blocker L-768,673, represents the first in vivo assessment of the cardiac electrophysiological and antiarrhythmic effects of selective I(Ks) blockade.

In an anesthetized canine model of recent (8.5+/-0.4 days) anterior myocardial infarction, 0.003 to 0.03 mg/kg L-768,673 IV significantly suppressed electrically induced ventricular tachyarrhythmias and reduced the incidence of lethal arrhythmias precipitated by acute, thrombotically induced posterolateral myocardial ischemia. Antiarrhythmic protection afforded by L-768,673 was accompanied by modest 7% to 10% increases in noninfarct zone ventricular effective refractory period, 3% to 5% increases in infarct zone ventricular effective refractory period, and 4% to 6% increases in QTc interval. In a conscious canine model of healed (3 to 4 weeks) anterior myocardial infarction, ventricular fibrillation was provoked by transient occlusion of the left circumflex coronary artery during submaximal exercise. Pretreatment with 0.03 mg/kg L-768,673 IV elicited a modest 7% increase in QTc, prevented ventricular fibrillation in 5 of 6 animals, and suppressed arrhythmias in 2 additional animals.

The present findings suggest that selective blockade of I(Ks) may be a potentially useful intervention for the prevention of malignant ischemic ventricular arrhythmias.

Our goal was to demonstrate the feasibility of an in vivo noninvasive method for imaging spinal cord cholinergic terminals using (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) and PET.

In vitro and in vivo experiments in rats were conducted to demonstrate the specific binding characteristics, localization, and time course of [3H]FBT binding in the spinal cord. PET imaging was then performed on seven rhesus monkeys.

The rat studies demonstrate high specific binding in the spinal cord with a distribution coinciding with the known distribution of cholinergic terminals. In vivo tracer concentrations in the spinal cord and basal ganglia were of the same magnitude. With use of [18F]FBT and PET in the rhesus monkey, the spinal cord was clearly visualized, with tracer concentration in the spinal cord being approximately one-fourth of that seen in the basal ganglia.

This work demonstrates the feasibility of imaging cholinergic terminals in vivo in the spinal cord using [18F]FBT and PET.