Attenuating persistent sodium current-induced atrial myopathy and fibrillation by preventing mitochondrial oxidative stress.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na+ current-induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

Ryanodine receptor remodeling in cardiomyopathy and muscular dystrophy caused by lamin A/C gene mutation.

Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause several diseases called laminopathies, the most common of which is dilated cardiomyopathy with muscular dystrophy. The role of Ca2+ regulation in these diseases remain poorly understood. We now show biochemical remodeling of the ryanodine receptor (RyR)/intracellular Ca2+ release channel in heart samples from human subjects with LMNA mutations, including protein kinase A-catalyzed phosphorylation, oxidation and depletion of the stabilizing subunit calstabin. In the LmnaH222P/H222P murine model of Emery-Dreifuss muscular dystrophy caused by LMNA mutation, we demonstrate an age-dependent biochemical remodeling of RyR2 in the heart and RyR1 in skeletal muscle. This RyR remodeling is associated with heart and skeletal muscle dysfunction. Defective heart and muscle function are ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes 'leaky' RyRs. SMAD3 phosphorylation is increased in hearts and diaphragms of LmnaH222P/H222P mice, which enhances NADPH oxidase binding to RyR channels, contributing to their oxidation. There is also increased generalized protein oxidation, increased calcium/calmodulin-dependent protein kinase II-catalyzed phosphorylation of RyRs and increased protein kinase A activity in these tissues. Our data show that RyR remodeling plays a role in cardiomyopathy and skeletal muscle dysfunction caused by LMNA mutation and identify these Ca2+ channels as a potential therapeutic target.

Dietary Saturated Fat Promotes Arrhythmia by Activating NOX2 (NADPH Oxidase 2).

BACKGROUND: Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism. METHODS: We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks. RESULTS: We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less inducible ventricular tachycardia. High saturated fat diet activates CaMK (Ca2+/calmodulin-dependent protein kinase) in the heart, which contributes to abnormal calcium handling, promoting arrhythmia. CONCLUSIONS: We conclude that NOX2 deletion or pharmacological inhibition prevents the arrhythmogenic effects of a high saturated fat diet, in part mediated by activation of CaMK. This work reveals a molecular mechanism linking cardiac metabolism to arrhythmia and suggests that NOX2 inhibitors could be a novel therapy for heart rhythm abnormalities caused by cardiac lipid overload.

Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure.

BACKGROUND: Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca2+ is released from the sarcoplasmic reticulum into the cytoplasm through type-2 ryanodine receptor/Ca2+ release channels. In CHF, chronically elevated circulating catecholamine levels cause pathological remodeling of type-2 ryanodine receptor/Ca2+ release channels resulting in diastolic sarcoplasmic reticulum Ca2+ leak and decreased myocardial contractility. Similarly, skeletal muscle contraction requires sarcoplasmic reticulum Ca2+ release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1-mediated sarcoplasmic reticulum Ca2+ leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine-responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca2+ handling because of leaky RyR channels in CHF. METHODS: Whole blood was collected from patients with CHF, CHF following left-ventricular assist device implant, and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF+S107 (a drug that specifically reduces RyR channel Ca2+ leak), and wild-type controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte-enriched preparations. RyR1 Ca2+ leak was assessed using flow cytometry to measure Ca2+ fluorescence in B-lymphocytes in the absence and presence of RyR1 agonists that empty RyR1 Ca2+ stores within the endoplasmic reticulum. RESULTS: Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased endoplasmic reticulum Ca2+ stores, consistent with chronic intracellular Ca2+ leak. This Ca2+ leak correlated with circulating catecholamine levels. The intracellular Ca2+ leak was significantly reduced in mice treated with the Rycal S107. Patients with CHF treated with left-ventricular assist devices exhibited a heterogeneous response. CONCLUSIONS: In CHF, B-lymphocytes exhibit remodeled leaky RyR1 channels and decreased endoplasmic reticulum Ca2+ stores consistent with chronic intracellular Ca2+ leak. RyR1-mediated Ca2+ leak in B-lymphocytes assessed using flow cytometry provides a surrogate measure of intracellular Ca2+ handling and systemic sympathetic burden, presenting a novel biomarker for monitoring response to pharmacological and mechanical CHF therapy.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with ß-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Maintenance of normal blood pressure is dependent on IP3R1-mediated regulation of eNOS.

Endothelial cells (ECs) are critical mediators of blood pressure (BP) regulation, primarily via the generation and release of vasorelaxants, including nitric oxide (NO). NO is produced in ECs by endothelial NO synthase (eNOS), which is activated by both calcium (Ca(2+))-dependent and independent pathways. Here, we report that intracellular Ca(2+) release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca(2+)-dependent eNOS activation. EC-specific type 1 1,4,5-trisphosphate receptor knockout (IP3R1(-/-)) mice are hypertensive and display blunted vasodilation in response to acetylcholine (ACh). Moreover, eNOS activity is reduced in both isolated IP3R1-deficient murine ECs and human ECs following IP3R1 knockdown. IP3R1 is upstream of calcineurin, a Ca(2+)/calmodulin-activated serine/threonine protein phosphatase. We show here that the calcineurin/nuclear factor of activated T cells (NFAT) pathway is less active and eNOS levels are decreased in IP3R1-deficient ECs. Furthermore, the calcineurin inhibitor cyclosporin A, whose use has been associated with the development of hypertension, reduces eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 plays a crucial role in the EC-mediated vasorelaxation and the maintenance of normal BP.

Mitochondrial calcium overload is a key determinant in heart failure.

Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation-contraction (E-C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF.

Mitochondrial oxidative stress promotes atrial fibrillation.

Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca(2+) release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca(2+) leak. Mice with intracellular Ca(2+) leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF.

Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging.

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.

Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.

Defective cardiac ryanodine receptor regulation during atrial fibrillation.

BACKGROUND: Ca2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. METHODS AND RESULTS: Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca2+ leak. CONCLUSIONS: SR Ca2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak.

BACKGROUND: Familial polymorphic ventricular tachycardia (FPVT) is characterized by exercise-induced arrhythmias and sudden cardiac death due to missense mutations in the cardiac ryanodine receptor (RyR2), an intracellular Ca2+ release channel required for excitation-contraction coupling in the heart. METHODS AND RESULTS: Three RyR2 missense mutations, P2328S, Q4201R, and V4653F, which occur in Finnish families, result in similar mortality rates of approximately 33% by age 35 years and a threshold heart rate of 130 bpm, above which exercise induces ventricular arrhythmias. Exercise activates the sympathetic nervous system, increasing cardiac performance as part of the fight-or-flight stress response. We simulated the effects of exercise on mutant RyR2 channels using protein kinase A (PKA) phosphorylation. All 3 RyR2 mutations exhibited decreased binding of calstabin2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. After PKA phosphorylation, FPVT-mutant RyR2 channels showed a significant gain-of-function defect consistent with leaky Ca2+ release channels and a significant rightward shift in the half-maximal inhibitory Mg2+ concentration (IC50). Treatment with the experimental drug JTV519 enhanced binding of calstabin2 to RyR2 and normalized channel function. CONCLUSIONS: Sympathetic activation during exercise induces ventricular arrhythmias above a threshold heart rate in RyR2 mutation carriers. Simulating the downstream effects of the sympathetic activation by PKA phosphorylation of RyR2 channels containing these FPVT missense mutations produced a consistent gain-of-function defect. RyR2 function and calstabin2 depletion were rescued by JTV519, suggesting stabilization of the RyR2 channel complex may represent a molecular target for the treatment and prevention of exercise-induced arrhythmias and sudden death in these patients.

Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2.

Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with normal hearts and in those with underlying disease such as heart failure. In animals with heart failure and in patients with inherited forms of exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2 (FKBP12.6) from the ryanodine receptor-calcium release channel (RyR2) complex causes an intracellular Ca2+ leak that can trigger fatal cardiac arrhythmias. A derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2 for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may be a therapeutic strategy for common ventricular arrhythmias.

Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor.

The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the "fight-or-flight" stress response. CaMKII phosphorylation increased RyR2 Ca2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca2+-induced Ca2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org.

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.