Efficacy and potency of class I antiarrhythmic drugs for suppression of Ca2+ waves in permeabilized myocytes lacking calsequestrin

Ca²⁺ waves can trigger ventricular arrhythmias such as catecholaminergic–polymorphic ventricular tachycardia (CPVT). Drugs that prevent Ca²⁺ waves may have antiarrhythmic properties. Here, we use permeabilized ventricular myocytes from a CPVT mouse model lacking calsequestrin (casq2) to screen all clinically available class I antiarrhythmic drugs and selected other antiarrhythmic agents for activity against Ca²⁺ waves. Casq2−/− myocytes were imaged in line-scan mode and the following Ca²⁺ wave parameters analyzed: wave incidence, amplitude, frequency, and propagation speed. IC₅₀ (potency) and maximum inhibition (efficacy) were calculated for each drug. Drugs fell into 3 distinct categories. Category 1 drugs (flecainide and R-propafenone) suppressed wave parameters with the highest potency (IC₅₀ < 10 μM) and efficacy (> 50% maximum wave inhibition). Category 2 drugs (encainide, quinidine, lidocaine, and verapamil) had intermediate potency (IC₅₀ 20–40 μM) and efficacy (20–40% maximum wave inhibition). Category 3 drugs (procainamide, disopyramide, mexiletine, cibenzoline, and ranolazine) had no significant effects on Ca²⁺ waves at the highest concentration tested (100 μM). Propafenone was stereoselective, with R-propafenone suppressing waves more potently than S-propafenone (IC₅₀: R-propafenone 2 ± 0.2 μM vs. S-propafenone 54 ± 18 μM). Both flecainide and R-propafenone decreased Ca²⁺ spark mass and converted propagated Ca²⁺ waves into non-propagated wavelets and frequent sparks, suggesting that reduction in spark mass, not spark frequency, was responsible for wave suppression. Among all class I antiarrhythmic drugs, flecainide and R-propafenone inhibit Ca²⁺ waves with the highest potency and efficacy. Permeabilized casq2−/− myocytes are a simple in-vitro assay for finding drugs with activity against Ca²⁺ waves. This article is part of a Special Issue entitled ‘Possible Editorial’.     

Prevention of Ventricular Arrhythmia and Calcium Dysregulation in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin‐2 Mutation

Arrhythmia Prevention in CPVT . Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium‐regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin‐2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca²⁺ overload and increased Ca²⁺ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. Method and results: CPVT mouse models that lack CASQ2 were treated with Ca²⁺‐channel inhibitors, β‐adrenergic inhibitors, or Mg²⁺. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca²⁺ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress‐induced ventricular arrhythmia in mutant mice. The Ca²⁺ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca²⁺ content in mutant myocytes, diminished diastolic Ca²⁺ overload, increased systolic Ca²⁺ amplitude, and prevented Ca²⁺ oscillations in stressed mutant myocytes. Conclusions: Ca²⁺ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil‐induced partial normalization of SR Ca²⁺ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca²⁺ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT . (J Cardiovasc Electrophysiol, Vol. 22, pp. 316‐324, March 2011)     

Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.     

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.     

Role of CaMKII in RyR leak, EC coupling and action potential duration: A computational model

During heart failure, the ability of the sarcoplasmic reticulum (SR) to store Ca²⁺ is severely impaired resulting in abnormal Ca²⁺ cycling and excitation-contraction (EC) coupling. Recently, it has been proposed that “leaky” ryanodine receptors (RyRs) contribute to diminished Ca²⁺ levels in the SR. Various groups have experimentally investigated the effects of RyR phosphorylation mediated by Ca²⁺/calmodulin-dependent kinase II (CaMKII) on RyR behavior. Some of these results are difficult to interpret since RyR gating is modulated by many external proteins and ions, including Ca²⁺. Here, we present a mathematical model representing CaMKII-RyR interaction in the canine ventricular myocyte. This is an extension of our previous model which characterized CaMKII phosphorylation of L-type Ca²⁺ channels (LCCs) in the cardiac dyad. In this model, it is assumed that upon phosphorylation, RyR Ca²⁺-sensitivity is increased. Individual RyR phosphorylation is modeled as a function of dyadic CaMKII activity, which is modulated by local Ca²⁺ levels. The model is constrained by experimental measurements of Ca²⁺ spark frequency and steady state RyR phosphorylation. It replicates steady state RyR (leak) fluxes in the range measured in experiments without the addition of a separate passive leak pathway. Simulation results suggest that under physiological conditions, CaMKII phosphorylation of LCCs ultimately has a greater effect on RyR flux as compared with RyR phosphorylation. We also show that phosphorylation of LCCs decreases EC coupling gain significantly and increases action potential duration. These results suggest that LCC phosphorylation sites may be a more effective target than RyR sites in modulating diastolic RyR flux.     

High-mobility group box 1 (HMGB1) impaired cardiac excitation–contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca2 + leak through TLR4–ROS signaling in cardiomyocytes

High-mobility group box 1 (HMGB1) is a proinflammatory mediator playing an important role in the pathogenesis of cardiac dysfunction in many diseases. In this study, we explored the effects of HMGB1 on Ca^2 + handling and cellular contractility in cardiomyocytes to seek for the mechanisms underlying HMGB1-induced cardiac dysfunction. Our results show that HMGB1 increased the frequency of Ca^2 + sparks, reduced the sarcoplasmic reticulum (SR) Ca^2 + content, and decreased the amplitude of systolic Ca^2 + transient and myocyte contractility in dose-dependent manners in adult rat ventricular myocytes. Inhibiting high-frequent Ca^2 + sparks with tetracaine largely inhibited the alterations of SR load and Ca^2 + transient. Blocking Toll-like receptor 4 (TLR4) with TAK-242 or knockdown of TLR4 by RNA interference remarkably inhibited HMGB1 induced high-frequent Ca^2 + sparks and restored the SR Ca^2 + content. Concomitantly, the amplitude of systolic Ca^2 + transient and myocyte contractility had significantly increased. Furthermore, HMGB1 increased the level of intracellular reactive oxygen species (ROS) and consequently enhanced oxidative stress and CaMKII-activated phosphorylation (pSer2814) in ryanodine receptor 2 (RyR2). TAK-242 pretreatment significantly decreased intracellular ROS levels and oxidative stress and hyperphosphorylation in RyR2, similar to the effects of antioxidant MnTBAP. Consistently, MnTBAP normalized HMGB1-impaired Ca^2 + handling and myocyte contractility. Taken together, our findings suggest that HMGB1 enhances Ca^2 + spark-mediated SR Ca^2 + leak through TLR4–ROS signaling pathway, which causes partial depletion of SR Ca^2 + content and hence decreases systolic Ca^2 + transient and myocyte contractility. Prevention of SR Ca^2 + leak may be an effective therapeutic strategy for the treatment of cardiac dysfunction related to HMGB1 overproduction.     

Calmodulin kinase II inhibition prevents arrhythmias in RyR2(R4496C+/-) mice with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by life-threatening arrhythmias elicited by adrenergic activation. CPVT is caused by mutations in the cardiac ryanodine receptor gene (RyR2). In vitro studies demonstrated that RyR2 mutations respond to sympathetic activation with an abnormal diastolic Ca(2+) leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2(R4496C+/-) knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) counteracts the effects of adrenergic stimulation resulting in an antiarrhythmic activity. CaMKII inhibition with KN-93 completely prevented catecholamine-induced sustained ventricular tachyarrhythmia in RyR2(R4496C+/-) mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2(R4496C+/-) mice, CaMKII inhibition with an autocamtide-2 related inhibitory peptide or with KN-93 blunted triggered activity and transient inward currents induced by isoproterenol. Isoproterenol also enhanced the activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), increased spontaneous Ca(2+) release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca(2+) content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2(R4496C+/-) knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca(2+) leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy

Aberrant intracellular Ca²⁺ regulation is believed to contribute to the development of cardiomyopathy in Duchenne muscular dystrophy. Here, we tested whether inhibition of protein kinase A (PKA) phosphorylation of ryanodine receptor type 2 (RyR2) prevents dystrophic cardiomyopathy by reducing SR Ca²⁺ leak in the mdx mouse model of Duchenne muscular dystrophy. mdx mice were crossed with RyR2-S2808A mice, in which PKA phosphorylation site S2808 on RyR2 is inactivated by alanine substitution. Compared with mdx mice that developed age-dependent heart failure, mdx-S2808A mice exhibited improved fractional shortening and reduced cardiac dilation. Whereas application of isoproterenol severely depressed cardiac contractility and caused 95% mortality in mdx mice, contractility was preserved with only 19% mortality in mdx-S2808A mice. SR Ca²⁺ leak was greater in ventricular myocytes from mdx than mdx-S2808A mice. Myocytes from mdx mice had a higher incidence of isoproterenol-induced diastolic Ca²⁺ release events than myocytes from mdx-S2808A mice. Thus, inhibition of PKA phosphorylation of RyR2 reduced SR Ca²⁺ leak and attenuated cardiomyopathy in mdx mice, suggesting that enhanced PKA phosphorylation of RyR2 at S2808 contributes to abnormal Ca²⁺ homeostasis associated with dystrophic cardiomyopathy.     

Calcium leak through ryanodine receptors leads to atrial fibrillation in 3 mouse models of catecholaminergic polymorphic ventricular tachycardia

Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca(2+)) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective: In the present study, we sought to determine whether intracellular diastolic SR Ca(2+) leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results: We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca(2+) leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S(+/-), 70%; RyR2-N2386I(+/-), 60%; RyR2-L433P(+/-), 35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca(2+) leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca(2+) leak through the channel. Atrial RyR2 from RyR2-R2474S(+/-) mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca(2+) leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions: The present study demonstrates that RyR2-mediated diastolic SR Ca(2+) leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca(2+) leak through RyR2 and pacing-induced AF associated with CPVT mutations.     

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter,RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H₂O₂)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H₂O₂ was used to mimic oxidative stress. H₂O₂ significantly increased the frequency of Ca^2 + sparks and spontaneous Ca^2 + waves, and dantrolene almost completely blocked these effects. H₂O₂ pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H₂O₂ also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H₂O₂ causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H₂O₂ treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

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) Ca²⁺ 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 Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ 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 Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure

Altered sarcoplasmic reticulum (SR) Ca2+-ATPase and Na+-Ca2+ exchange (NCX) function have been implicated in depressing SR Ca2+ content and contractile function in heart failure (HF). Enhanced diastolic ryanodine receptor (RyR) leak could also lower SR Ca2+ load in HF, but direct cellular measurements are lacking. In this study, we measure SR Ca2+ leak directly in intact isolated rabbit ventricular myocytes from a well-developed nonischemic HF model. Abrupt block of SR Ca2+ leak by tetracaine shifts Ca2+ from the cytosol to SR. The tetracaine-induced decline in [Ca2+]i and increase total SR Ca2+ load ([Ca2+]SRT) directly indicate the SR Ca2+ leak (before tetracaine). Diastolic SR Ca2+ leak increases with [Ca2+]SRT, and for any [Ca2+]SRT is greater in HF versus control. Mathematical modeling was used to compare the relative impact of alterations in SR Ca2+ leak, SR Ca2+-ATPase, and Na+-Ca2+ exchange on SR Ca2+ load in HF. We conclude that increased diastolic SR Ca2+ leak in HF may contribute to reductions in SR Ca2+ content, but changes in NCX in this HF model have more impact on [Ca2+]SRT.     

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca²⁺ 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 Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ 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 Ca²⁺/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 [Ca²⁺]_i and oxidative stress), and that therapies restoring nitroso–redox balance in the heart could prevent sudden arrhythmic death.Nitric oxide (NO) is an important regulator of cardiac function via both the activation of cyclic guanosine monophosphate-dependent signaling pathways and direct posttranslational modification of protein thiols (S-nitrosylation) (1). NO derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins (2). In particular, nitrosylation of both skeletal and cardiac muscle ryanodine receptors (RyR1 and RyR2, respectively) alters their release properties, favoring activation (3, 4). Notably, an increase in RyR2 open probability can cause spontaneous SR Ca²⁺ release, which may cause arrhythmias. Recently, it was shown that decreased RyR2 S-nitrosylation (RyR2-SNO) through loss of NOS1, was associated with increased spontaneous SR Ca²⁺ release events in isolated cardiomyocytes, following rapid pacing (5). In a separate study, NOS1 deficiency was shown to decrease spontaneous SR Ca²⁺ sparks and the open probability of RyR2 under resting conditions in cardiomyocytes and lipid bilayers, respectively (6). These studies suggest that NOS1 deficiency has a variable effect on RyR2 function, likely dependent on the underlying physiological state (i.e., rapid heart rate versus quiescence). It remains unknown, however, whether these changes create a substrate for arrhythmogenesis in the intact heart.It is increasingly evident that activities of nitric oxide and reactive oxygen species (ROS) are tightly coupled in cardiomyocytes producing nitroso–redox balance. Elevated ROS production (oxidative stress) is a hallmark of several cardiovascular diseases associated with increased risk of fatal ventricular arrhythmias [e.g., myocardial infarction (MI) and heart failure]. Burger et al. (7) recently demonstrated an increased incidence of ventricular arrhythmias following MI in NOS1-deficient mice. These data suggest that a nitroso–redox imbalance may be arrhythmogenic in the setting of MI. However, the molecular basis of the increased arrhythmogenesis is not known.In the current study, we found that decreased NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release resulting from a combination of decreased RyR2-SNO and increased RyR2 oxidation (RyR2-SOx) [i.e., increased ROS from xanthine oxidoreductase (XOR) activity] and could be suppressed with xanthine oxidoreductase inhibition (i.e., allopurinol) or nitric oxide donors (i.e., GSNO). Notably, we found evidence of NOS1 regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between the nitrosylation and phosphorylation states of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype under oxidative stress.     

Excitation–contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca²⁺ with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca²⁺ transient due to reduced ability of the l-type Ca²⁺ channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca²⁺, with some cellular regions failing to elicit Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca²⁺ sparks. Monte-Carlo simulations of spark activation by l-type Ca²⁺ current supported the idea that the decreased synchrony of Ca²⁺ spark production associated with the loss of the notch could be explained by reduced Ca²⁺ influx from open Ca²⁺ channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca²⁺ release in heart failure, without changes in (for example) SR Ca²⁺-ATPase uptake.     

Ca2 + Sparks and Ca2 + waves are the subcellular events underlying Ca2 + overload during ischemia and reperfusion in perfused intact hearts

Abnormal intracellular Ca²⁺ cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca²⁺. At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca^2+ +, which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca²⁺ increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses:  (1) An increase diastolic Ca²⁺ sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca²⁺ increase; (2) an increase in cytosolic Ca²⁺ pro-arrhythmogenic events (Ca²⁺ waves), mediates the abrupt diastolic Ca²⁺ rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca²⁺ sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07 ± 0.33 vs. 1.13 ± 0.20 sp/s/100 μm, n = 29/34, 7 hearts). Reperfusion significantly changed Ca²⁺ sparks kinetics, by prolonging Ca²⁺ sparks rise time and decreased cspf. However, it significantly increased Ca²⁺ wave frequency relative to ischemia (0.71 ± 0.14 vs. 0.38 ± 0.06 w/s/100 μm, n = 32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca^2 + sparks and provides direct evidence of increased Ca²⁺ sparks in ischemia that transform into Ca²⁺ waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.     

Excitation–contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca²⁺ with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca²⁺ transient due to reduced ability of the l-type Ca²⁺ channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca²⁺, with some cellular regions failing to elicit Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca²⁺ sparks. Monte-Carlo simulations of spark activation by l-type Ca²⁺ current supported the idea that the decreased synchrony of Ca²⁺ spark production associated with the loss of the notch could be explained by reduced Ca²⁺ influx from open Ca²⁺ channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca²⁺ release in heart failure, without changes in (for example) SR Ca²⁺-ATPase uptake.     

Ryanodin-Rezeptor-Stabilisatoren

Different disease syndromes including arrhythmias, heart failure, skeletal myopathy, and epilepsy have been associated with abnormally increased Ca²⁺ leak from the intracellular organelles. In the heart, intracellular Ca²⁺ release is controlled by cardiac ryanodine receptors (RyR2s) which are Ca²⁺ release channels situated in the membranes of the sarcoplasmic reticulum (SR) storage organelles. RyR2-dependent Ca²⁺ release is essential for myocardial contraction and relaxation which control systolic and diastolic heart function. The function of the Ca²⁺ release channel depends on four identical RyR2 subunits each associated with a specific set of enzymes which modulate cardiac function. Both genetic and acquired forms of heart disease result in unstable RyR2 channel closure in diastole and detrimental SR Ca²⁺ leak. The acute form of SR Ca²⁺ leak leads to afterdepolarizations of the membrane potential which may trigger deadly arrhythmias, whereas the chronic form of SR Ca²⁺ leak depletes intracellular Ca²⁺ stores and contributes to heart failure. Leaky RyR2 channels are the pharmacological target of novel RyR-selective 1,4-benzothiazepin derivatives, which were found to stabilize the channel closed state through increased calstabin2 binding, to inhibit SR Ca²⁺ leak, and to exert therapeutic in vivo effects against arrhythmias and heart failure progression.     

Role and mechanism of subcellular Ca2+ distribution in the action of two inotropic agents with different toxicity

Pro-arrhythmic risk strongly limits the therapeutic value of current inotropic interventions. Istaroxime (previously PST2744) is a novel inotropic agent, significantly less pro-arrhythmic than digoxin that, in addition to block Na⁺/K⁺ pump, stimulates sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2). Here we compare istaroxime and digoxin effects to further address the role of SR modulation in reducing the toxicity associated with Na⁺/K⁺ pump blockade. In murine ventricular myocytes both compounds increased cell twitch (inotropy) in a concentration-dependent fashion. At high concentrations digoxin, but not istaroxime, induced unstimulated contractions, a sign of pro-arrhythmic toxicity. To evaluate the mechanism of this difference, we compared the two drugs at concentrations exerting equal inotropy but different toxicity. At these concentrations: (1) the two drugs equally inhibited the Na⁺/K⁺ pump; (2) digoxin induced larger increases in resting Ca²⁺ and in diastolic Ca²⁺ during pacing; (3) neither drug affected the relationship between RyR-mediated SR Ca²⁺ leak and Ca²⁺ content; (4) istaroxime, but not digoxin, enhanced SR Ca²⁺ reuptake rate. In conclusion, digoxin toxicity was associated to larger accumulation of cytosolic Ca²⁺, which did not result from RyR facilitation, but which might ultimately induce it to promote unstimulated Ca²⁺ release. The lower toxicity of Na⁺/K⁺ pump blockade by istaroxime may thus reflect improved Ca²⁺ confinement within the SR, likely to result from concomitant SERCA2 stimulation.     

Mechanism underlying catecholaminergic polymorphic ventricular tachycardia and approaches to therapy

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by VT induced by adrenergic stress in the absence of structural heart disease and high incidence of sudden cardiac death. The diagnosis is made based on reproducible ventricular tachyarrhythmias including bidirectional VT and polymorphic VT during exercise testings. Two causative genes of CPVT have been identified: RYR2, encoding the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel, and CASQ2, encoding cardiac calsequestrin. A mutation in RYR2 or CASQ2 is identified in approximately 60% of patients with CPVT. Mutations in these two genes destabilize the RyR2 Ca²⁺ release channel complex in sarcoplasmic reticulum and result in spontaneous Ca²⁺ release through RyR2 channels leading to delayed after depolarization, triggered activity, and bidirectional/polymorphic VT. Implantable cardioverter defibrillators (ICDs) are recommended for prevention of sudden death in patients with CPVT.1. A.E. Epstein, J.P. DiMarco, K.A. Ellenbogen, et al., ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices): developed in collaboration with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Circulation. 2008;117:e350 However, painful shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality, but is not completely effective. The beneficial effects of Ca²⁺ channel blocker verapamil in combination with β-blocker have been reported, but the role of verapamil has not been well assessed. Because Ca²⁺ leakage through ryanodine channel is a common mechanism of CPVT, ryanodine channel block may have a therapeutic effect. We discovered that flecainide directly inhibits RyR2 channels and prevent CPVT. Left cardiac sympathetic denervation may be an effective alternative treatment in combination with ICD, especially for patients whose arrhythmias are not controlled by drug therapies.