STIM1 ablation impairs exercise-induced physiological cardiac hypertrophy and dysregulates autophagy in mouse hearts.

Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.

MATE1 Deficiency Exacerbates Dofetilide-Induced Proarrhythmia.

Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS.

Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under ß-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.

Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated IKado.

AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 µM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p < 0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SIGNIFICANCE: An increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease.

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts.

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of ß-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMKII suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMKII- (and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF.

In Utero Particulate Matter Exposure Produces Heart Failure, Electrical Remodeling, and Epigenetic Changes at Adulthood.

BACKGROUND: Particulate matter (PM; PM2.5 [PM with diameters of <2.5 µm]) exposure during development is strongly associated with adverse cardiovascular outcomes at adulthood. In the present study, we tested the hypothesis that in utero PM2.5 exposure alone could alter cardiac structure and function at adulthood. METHODS AND RESULTS: Female FVB mice were exposed either to filtered air or PM2.5 at an average concentration of 73.61 µg/m3 for 6 h/day, 7 days/week throughout pregnancy. After birth, animals were analyzed at 12 weeks of age. Echocardiographic (n=9-10 mice/group) and pressure-volume loop analyses (n=5 mice/group) revealed reduced fractional shortening, increased left ventricular end-systolic and -diastolic diameters, reduced left ventricular posterior wall thickness, end-systolic elastance, contractile reserve (dP/dtmax/end-systolic volume), frequency-dependent acceleration of relaxation), and blunted contractile response to ß-adrenergic stimulation in PM2.5-exposed mice. Isolated cardiomyocyte (n=4-5 mice/group) function illustrated reduced peak shortening, ±dL/dT, and prolonged action potential duration at 90% repolarization. Histological left ventricular analyses (n=3 mice/group) showed increased collagen deposition in in utero PM2.5-exposed mice at adulthood. Cardiac interleukin (IL)-6, IL-1ß, collagen-1, matrix metalloproteinase (MMP) 9, and MMP13 gene expressions were increased at birth in in utero PM2.5-exposed mice (n=4 mice/group). In adult hearts (n=5 mice/group), gene expressions of sirtuin (Sirt) 1 and Sirt2 were decreased, DNA methyltransferase (Dnmt) 1, Dnmt3a, and Dnmt3b were increased, and protein expression (n=6 mice/group) of Ca2+-ATPase, phosphorylated phospholamban, and Na+/Ca2+ exchanger were decreased. CONCLUSIONS: In utero PM2.5 exposure triggers an acute inflammatory response, chronic matrix remodeling, and alterations in Ca2+ handling proteins, resulting in global adult cardiac dysfunction. These results also highlight the potential involvement of epigenetics in priming of adult cardiac disease.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.

Chronic Omega-3 Polyunsaturated Fatty Acid Treatment Variably Affects Cellular Repolarization in a Healed Post-MI Arrhythmia Model.

INTRODUCTION: Over the last 40 years omega-3 polyunsaturated fatty acids (PUFAs) have been shown to be anti-arrhythmic or pro-arrhythmic depending on the method and duration of administration and model studied. We previously reported that omega-3 PUFAs do not confer anti-arrhythmic properties and are pro-arrhythmic in canine model of sudden cardiac death (SCD). Here, we evaluated the effects of chronic omega-3 PUFA treatment in post-MI animals susceptible (VF+) or resistant (VF-) to ventricular tachyarrhythmias. METHODS: Perforated patch clamp techniques were used to measure cardiomyocyte action potential durations (APD) at 50 and 90% repolarization and short term variability of repolarization. The early repolarizing transient outward potassium current Ito was also studied. RESULTS: Omega-3 PUFAs prolonged the action potential in VF- myocytes at both 50 and 90% repolarization. Short term variability of repolarization was increased in both untreated and treated VF- myocytes vs. CONTROLS: Ito was unaffected by omega-3 PUFA treatment. Omega-3 PUFA treatment attenuated the action potential prolongation in VF+ myocytes, but did not return repolarization to control values. CONCLUSIONS: Omega-3 PUFAs do not confer anti-arrhythmic properties in the setting of healed myocardial infarction in a canine model of SCD. In canines previously resistant to ventricular fibrillation (VF-), omega-3 PUFA treatment prolonged the action potential in VF- myocytes, and may contribute to pro-arrhythmic responses.

Dysfunction in the ßII spectrin-dependent cytoskeleton underlies human arrhythmia.

BACKGROUND: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. METHODS AND RESULTS: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that ßII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. ßII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/ßII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac ßII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, ßII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in ßII spectrin-deficient mice. CONCLUSIONS: Our findings identify ßII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

Heart failure duration progressively modulates the arrhythmia substrate through structural and electrical remodeling.

AIMS: Ventricular arrhythmias are a common cause of death in patients with heart failure (HF). Structural and electrical abnormalities in the heart provide a substrate for such arrhythmias. Canine tachypacing-induced HF models of 4-6 weeks duration are often used to study pathophysiology and therapies for HF. We hypothesized that a chronic canine model of HF would result in greater electrical and structural remodeling than a short term model, leading to a more arrhythmogenic substrate. MAIN METHODS: HF was induced by ventricular tachypacing for one (short-term) or four (chronic) months to study remodeling. KEY FINDINGS: Left ventricular contractility was progressively reduced, while ventricular hypertrophy and interstitial fibrosis were evident at 4 month but not 1 month of HF. Left ventricular myocyte action potentials were prolonged after 4 (p<0.05) but not 1 month of HF. Repolarization instability and early afterdepolarizations were evident only after 4 months of HF (p<0.05), coinciding with a prolonged QTc interval (p<0.05). The transient outward potassium current was reduced in both HF groups (p<0.05). The outward component of the inward rectifier potassium current was reduced only in the 4 month HF group (p<0.05). The delayed rectifier potassium currents were reduced in 4 (p<0.05) but not 1 month of HF. Reactive oxygen species were increased at both 1 and 4 months of HF (p<0.05). SIGNIFICANCE: Reduced Ito, outward IK1, IKs, and IKr in HF contribute to EAD formation. Chronic, but not short term canine HF, results in the altered electrophysiology and repolarization instability characteristic of end-stage human HF.

Calcium-activated potassium current modulates ventricular repolarization in chronic heart failure.

The role of I(KCa) in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of I(KCa) blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The I(KCa) blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by I(KCa) blockade in all of the groups studied. I(KCa) blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. I(KCa) blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, I(KCa) blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for I(KCa) in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.

Ibandronate and ventricular arrhythmia risk.

INTRODUCTION: Bisphosphonates, including ibandronate, are used in the prevention and treatment of osteoporosis. METHODS AND RESULTS: We report a case of suspected ibandronate-associated arrhythmia, following a single dose of ibandronate in a 55-year-old female. ECG at presentation revealed frequent ectopy and QT/QTc interval prolongation; at follow-up 9 months later the QT/QTc intervals were normalized. Proarrhythmic potential of ibandronate was assessed with a combination of in vivo and in vitro approaches in canines and canine ventricular myocytes. We observed late onset in vivo repolarization instability after ibandronate treatment. Myocytes superfused with ibandronate exhibited action potential duration (APD) prolongation and variability, increased early afterdepolarizations (EADs) and reduced Ito (P < 0.05), with no change in IKr . Ibandronate-induced APD changes and EADs were prevented by inhibition of intracellular calcium cycling. Ibandronate increased sarcoplasmic reticulum calcium load; during washout there was an increase in calcium spark frequency and spontaneous calcium waves. Computational modeling was used to examine the observed effects of ibandronate. While reductions in Ito alone had modest effects on APD, when combined with altered RyR inactivation kinetics, the model predicted effects on APD and SR Ca(2+) load consistent with observed experimental results. CONCLUSION: Ibandronate may increase the susceptibility to ventricular ectopy and arrhythmias. Collectively these data suggest that reduced Ito combined with abnormal RyR calcium handling may result in a previously unrecognized form of drug-induced proarrhythmia.

Dietary omega-3 fatty acids promote arrhythmogenic remodeling of cellular Ca2+ handling in a postinfarction model of sudden cardiac death.

It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca(2+) handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca(2+) cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca(2+) imaging techniques, we examined the intracellular Ca(2+) handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca(2+) waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca(2+) transients (Ca(2+) alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca(2+) spark frequency and reduced sarcoplasmic reticulum Ca(2+) content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca(2+) cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca(2+) handling.

Differential effects of the peroxynitrite donor, SIN-1, on atrial and ventricular myocyte electrophysiology.

Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), a peroxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole-cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate independent, whereas in the ventricle APD, prolongation was rate dependent. In addition to prolongation of the action potential, beat-to-beat variability of repolarization was significantly increased in ventricular but not in atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1-induced increase in ventricular beat-to-beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.

Endurance exercise training normalizes repolarization and calcium-handling abnormalities, preventing ventricular fibrillation in a model of sudden cardiac death.

The risk of sudden cardiac death is increased following myocardial infarction. Exercise training reduces arrhythmia susceptibility, but the mechanism is unknown. We used a canine model of sudden cardiac death (healed infarction, with ventricular tachyarrhythmias induced by an exercise plus ischemia test, VF+); we previously reported that endurance exercise training was antiarrhythmic in this model (Billman GE. Am J Physiol Heart Circ Physiol 297: H1171-H1193, 2009). A total of 41 VF+ animals were studied, after random assignment to 10 wk of endurance exercise training (EET; n = 21) or a matched sedentary period (n = 20). Following (>1 wk) the final attempted arrhythmia induction, isolated myocytes were used to test the hypotheses that the endurance exercise-induced antiarrhythmic effects resulted from normalization of cellular electrophysiology and/or normalization of calcium handling. EET prevented VF and shortened in vivo repolarization (P < 0.05). EET normalized action potential duration and variability compared with the sedentary group. EET resulted in a further decrement in transient outward current compared with the sedentary VF+ group (P < 0.05). Sedentary VF+ dogs had a significant reduction in repolarizing K(+) current, which was restored by exercise training (P < 0.05). Compared with controls, myocytes from the sedentary VF+ group displayed calcium alternans, increased calcium spark frequency, and increased phosphorylation of S2814 on ryanodine receptor 2. These abnormalities in intracellular calcium handling were attenuated by exercise training (P < 0.05). Exercise training prevented ischemically induced VF, in association with a combination of beneficial effects on cellular electrophysiology and calcium handling.

Prolonged Action Potential and After depolarizations Are Not due to Changes in Potassium Currents in NOS3 Knockout Ventricular Myocytes.

Ventricular myocytes deficient in endothelial nitric oxide synthase (NOS3(-/-)) exhibit prolonged action potential (AP) duration and enhanced spontaneous activity (early and delayed afterdepolarizations) during ß-adrenergic (ß-AR) stimulation. Studies have shown that nitric oxide is able to regulate various K(+) channels. Our objective was to examine if NOS3(-/-) myocytes had altered K(+) currents. APs, transient outward (I(to)), sustained (I(Ksus)), and inward rectifier (I(K1)) K(+) currents were measured in NOS3(-/-) and wild-type (WT) myocytes. During ß-AR stimulation, AP duration (measured as 90% repolarization-APD(90)) was prolonged in NOS3(-/-) compared to WT myocytes. Nevertheless, we did not observe differences in I(to), I(Ksus), or I(K1) between WT and NOS3(-/-) myocytes. Our previous work showed that NOS3(-/-) myocytes had a greater Ca(2+) influx via L-type Ca(2+) channels with ß-AR stimulation. Thus, we measured ß-AR-stimulated SR Ca(2+) load and found a greater increase in NOS3(-/-) versus WT myocytes. Hence, our data suggest that the prolonged AP in NOS3(-/-) myocytes is not due to changes in I(to), I(Ksus), or I(K1). Furthermore, the increase in spontaneous activity in NOS3(-/-) myocytes may be due to a greater increase in SR Ca(2+) load. This may have important implications for heart failure patients, where arrhythmias are increased and NOS3 expression is decreased.

Nitric oxide synthases and atrial fibrillation.

Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases (NOS), which normally produce nitric oxide in the heart. Two NOS isoforms (1 and 3) are normally expressed in the heart. During pathologies such as heart failure, there is induction of NOS 2 in multiple cell types in the myocardium. In certain conditions, the NOS enzymes may become uncoupled, shifting from production of nitric oxide to superoxide anion, a potent free radical and oxidant. Multiple lines of evidence suggest a role for NOS in the pathogenesis of atrial fibrillation. Therapeutic approaches to reduce atrial fibrillation by modulation of NOS activity may be beneficial, although further investigation of this strategy is needed.

Differential regulation of EHD3 in human and mammalian heart failure.

Electrical and structural remodeling during the progression of cardiovascular disease is associated with adverse outcomes subjecting affected patients to overt heart failure (HF) and/or sudden death. Dysfunction in integral membrane protein trafficking has long been linked with maladaptive electrical remodeling. However, little is known regarding the molecular identity or function of these intracellular targeting pathways in the heart. Eps15 homology domain-containing (EHD) gene products (EHD1-4) are polypeptides linked with endosomal trafficking, membrane protein recycling, and lipid homeostasis in a wide variety of cell types. EHD3 was recently established as a critical mediator of membrane protein trafficking in the heart. Here, we investigate the potential link between EHD3 function and heart disease. Using four different HF models including ischemic rat heart, pressure overloaded mouse heart, chronic pacing-induced canine heart, and non-ischemic failing human myocardium we provide the first evidence that EHD3 levels are consistently increased in HF. Notably, the expression of the Na/Ca exchanger (NCX1), targeted by EHD3 in heart is similarly elevated in HF. Finally, we identify a molecular pathway for EHD3 regulation in heart failure downstream of reactive oxygen species and angiotensin II signaling. Together, our new data identify EHD3 as a previously unrecognized component of the cardiac remodeling pathway.