Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart.
ABSTRACT: Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca(2+) handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca(2+) leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca(2+)-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca(2+)](i). These arrhythmias arose from increased spontaneous SR Ca(2+) release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca(2+)/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso-redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso-redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca(2+)](i) and oxidative stress), and that therapies restoring nitroso-redox balance in the heart could prevent sudden arrhythmic death.
Project description:S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca(2+) leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca(2+) leak, improving Ca(2+) handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca(2+) leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.
Project description:Altered Ca(2+) homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca(2+) homeostasis. Diastolic Ca(2+) levels are elevated in NOS1(-/-) and NOS1/NOS3(-/-) but not NOS3(-/-) myocytes compared with wild-type (WT), suggesting diastolic Ca(2+) leakage. Measured leak was increased in NOS1(-/-) and NOS1/NOS3(-/-) but not in NOS3(-/-) myocytes compared with WT. Importantly, NOS1(-/-) and NOS1/NOS3(-/-) myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1(-/-) hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca(2+) leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.
Project description: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-? 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-?-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.
Project description:Heart contraction vitally depends on tightly controlled intracellular Ca regulation. Because contraction is mainly driven by Ca released from the sarcoplasmic reticulum (SR), this organelle plays a particularly important role in Ca regulation. The type two ryanodine receptor (RyR2) is the major SR Ca release channel in ventricular myocytes. Several cardiac pathologies, including myocardial infarction and heart failure, are associated with increased RyR2 activity and diastolic SR Ca leak. It has been suggested that the increased RyR2 activity plays an important role in arrhythmias and contractile dysfunction. Several studies have linked increased SR Ca leak during myocardial infarction and heart failure to the activation of RyR2 in response to oxidative stress. This activation might include direct oxidation of RyR2 as well as indirect activation via phosphorylation or altered interactions with regulatory proteins. Out of ninety cysteine residues per RyR2 subunit, twenty one were reported to be in reduced state that could be potential targets for redox modifications that include S-nitrosylation, S-glutathionylation, and disulfide cross-linking. Despite its clinical significance, molecular mechanisms of RyR dysfunction during oxidative stress are not fully understood. Herein we review the most recent insights into redox-dependent modulation of RyR2 during oxidative stress and heart diseases.
Project description:Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in "leaky" RyR2 channels and a diastolic SR Ca(2+) leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca(2+) leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca(2+) leak prevents fatal sudden cardiac arrhythmias in DMD.
Project description:Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (?[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced ?[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 ?m), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.
Project description:Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects.To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak.We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-).Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca(2+).
Project description:?-Adrenergic receptor (?-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca(2+) signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca(2+) release that involves Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR).We tested whether physiological ?-AR activation causes Epac-mediated SR Ca(2+) leak and arrhythmias and whether it requires Epac1 versus Epac2, ?(1)-AR versus ?(2)-AR, and CaMKII?-dependent phosphorylation of RyR2-S2814. We used knockout (KO) mice for Epac1, Epac2, or both. All KOs exhibited unaltered basal cardiac function, Ca(2+) handling, and hypertrophy in response to pressure overload. However, SR Ca(2+) leak induced by the specific Epac activator 8-CPT in wild-type mice was abolished in Epac2-KO and double-KO mice but was unaltered in Epac1-KO mice. ?-AR-induced arrhythmias were also less inducible in Epac2-KO versus wild-type mice. ?-AR activation with protein kinase A inhibition mimicked 8-CPT effects on SR Ca(2+) leak and was prevented by blockade of ?(1)-AR but not ?(2)-AR. CaMKII inhibition (KN93) and genetic ablation of either CaMKII? or CaMKII phosphorylation on RyR2-S2814 prevented 8-CPT-induced SR Ca(2+) leak.?(1)-AR activates Epac2 to induce SR Ca(2+) leak via CaMKII?-dependent phosphorylation of RyR2-S2814. This pathway contributes to ?-AR-induced arrhythmias and reduced cardiac function.
Project description:Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ?-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.
Project description:In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF.Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca(2+)] in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of ?-adrenergic stimulation. In addition to causing arrhythmogenic-delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for the generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation.These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.