Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by adrenergically mediated polymorphic ventricular tachycardia leading to syncope and sudden cardiac death. The autosomal dominant form of CPVT is caused by mutations in the RyR2 gene encoding the cardiac isoform of the ryanodine receptor. In vitro functional characterization of mutant RyR2 channels showed altered behavior on adrenergic stimulation and caffeine administration with enhanced calcium release from the sarcoplasmic reticulum. As of today no experimental evidence is available to demonstrate that RyR2 mutations can reproduce the arrhythmias observed in CPVT patients. We developed a conditional knock-in mouse model carrier of the R4496C mutation, the mouse equivalent to the R4497C mutations identified in CPVT families, to evaluate if the animals would develop a CPVT phenotype and if beta blockers would prevent arrhythmias. Twenty-six mice (12 wild-type (WT) and 14RyR(R4496C)) underwent exercise stress testing followed by epinephrine administration: none of the WT developed ventricular tachycardia (VT) versus 5/14 RyR(R4496C) mice (P=0.02). Twenty-one mice (8 WT, 8 RyR(R4496C), and 5 RyR(R4496C) pretreated with beta-blockers) received epinephrine and caffeine: 4/8 (50%) RyR(R4496C) mice but none of the WT developed VT (P=0.02); 4/5 RyR(R4496C) mice pretreated with propranolol developed VT (P=0.56 nonsignificant versus RyR(R4496C) mice). These data provide the first experimental demonstration that the R4496C RyR2 mutation predisposes the murine heart to VT and VF in response caffeine and/or adrenergic stimulation. Furthermore, the results show that analogous to what is observed in patients, beta adrenergic stimulation seems ineffective in preventing life-threatening arrhythmias.     

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2(R4496C+/-)). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2(R4496C+/-) myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2(R4496C+/-) myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 micromol/L) but not by K201 (1 micromol/L or 10 micromol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2(R4496C+/-) mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2(R4496C+/-) either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2(R4496C+/-) myocytes and ventricular arrhythmias in RyR2(R4496C+/-) mice; and (3) RyR2-FKBP12.6 interaction in RyR2(R4496C+/-) is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.     

In Situ Confocal Imaging in Intact Heart Reveals Stress-Induced Ca2+ Release Variability in a Murine Catecholaminergic Polymorphic Ventricular Tachycardia Model of Type 2 Ryanodine ReceptorR4496C+/- Mutation

Background- Catecholaminergic polymorphic ventricular tachycardia is directly linked to mutations in proteins (eg, type 2 ryanodine receptor [RyR2](R4496C)) responsible for intracellular Ca(2+) homeostasis in the heart. However, the mechanism of Ca(2+) release dysfunction underlying catecholaminergic polymorphic ventricular tachycardia has only been investigated in isolated cells but not in the in situ undisrupted myocardium. Methods and Results- We investigated in situ myocyte Ca(2+) dynamics in intact Langendorff-perfused hearts (ex vivo) from wild-type and RyR2(R4496C+/-) mice using laser scanning confocal microscopy. We found that myocytes from both wild-type and RyR2(R4496C+/-) hearts displayed uniform, synchronized Ca(2+) transients. Ca(2+) transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in wild-type and RyR2(R4496C+/-) hearts, suggesting that excitation-contraction coupling between the sarcolemmal Ca(2+) channels and mutated RyR2(R4496C+/-) channels remains intact under baseline resting conditions. On adrenergic stimulation, RyR2(R4496C+/-) hearts exhibited a high degree of Ca(2+) release variability. The varied pattern of Ca(2+) release was absent in single isolated myocytes, independent of cell cycle length, synchronized among neighboring myocytes, and correlated with catecholaminergic polymorphic ventricular tachycardia. A similar pattern of action potential variability, which was synchronized among neighboring myocytes, was also revealed under adrenergic stress in intact hearts but not in isolated myocytes. Conclusions- Our studies using an in situ confocal imaging approach suggest that mutated RyR2s are functionally normal at rest but display a high degree of Ca(2+) release variability on intense adrenergic stimulation. Ca(2+) release variability is a Ca(2+) release abnormality, resulting from electric defects rather than the failure of the Ca(2+) release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca(2+) release and electric dysfunction in an established model of catecholaminergic polymorphic ventricular tachycardia.     

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.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).     

Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca2+ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugol's solution, and patch clamping in a knockin (RyR2/RyR2(R4496C)) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2(R4496C) and in 9% of 11 wild-type (P=0.03) littermates perfused with Ca2+and isoproterenol; 66% of 12 RyR2/RyR2(R4496C) and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (P=0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His-Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugol's solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2(R4496C) mice. Under current clamp, single Purkinje cells from RyR2/RyR2(R4496C) mouse hearts generated delayed afterdepolarization-induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His-Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT.     

Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death

Mutations in the human cardiac Ca2+ release channel (ryanodine receptor, RyR2) gene have recently been shown to cause effort-induced ventricular arrhythmias. However, the consequences of these disease-causing mutations in RyR2 channel function are unknown. In the present study, we characterized the properties of mutation R4496C of mouse RyR2, which is equivalent to a disease-causing human RyR2 mutation R4497C, by heterologous expression of the mutant in HEK293 cells. [3H]ryanodine binding studies revealed that the R4496C mutation resulted in an increase in RyR2 channel activity in particular at low Ca2+ concentrations. This increased basal channel activity remained sensitive to modulation by caffeine, ATP, Mg2+, and ruthenium red. In addition, the R4496C mutation enhanced the sensitivity of RyR2 to activation by Ca2+ and by caffeine. Single-channel analysis showed that single R4496C mutant channels exhibited considerable channel openings at low Ca2+ concentrations. HEK293 cells transfected with mutant R4496C displayed spontaneous Ca2+ oscillations more frequently than cells transfected with wild-type RyR2. Substitution of a negatively charged glutamate for the positively charged R4496 (R4496E) further enhanced the basal channel activity, whereas replacement of R4496 by a positively charged lysine (R4496K) had no significant effect on the basal activity. These observations indicate that the charge and polarity at residue 4496 plays an essential role in RyR2 channel gating. Enhanced basal activity of RyR2 may underlie an arrhythmogenic mechanism for effort-induced ventricular tachycardia.     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

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.     

Mutant ryanodine receptors in catecholaminergic polymorphic ventricular tachycardia generate delayed afterdepolarizations due to increased propensity to Ca2+ waves.

AIMS: Mutations in cardiac ryanodine receptors (RyR2s) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), characterized by risk of polymorphic ventricular tachyarrhythmias and sudden death during exercise. Arrhythmias are caused by gain-of-function defects in RyR2, but cellular arrhythmogenesis remains elusive. METHODS AND RESULTS: We recorded endocardial monophasic action potentials (MAPs) at right ventricular septum in 15 CPVT patients with a RyR2 mutation (P2,328S, Q4,201R, and V4,653F) and in 12 control subjects both at baseline and during epinephrine infusion (0.05 microg/kg/min). At baseline 3 and during epinephrine infusion, four CPVT patients, but none of the control subjects, showed delayed afterdepolarizations (DADs) occasionally coinciding with ventricular premature complexes. In order to study the underlying mechanisms, we expressed two types of mutant RyR2 (P2,328S and V4,653F) causing CPVT as well as wild-type RyR2 in HEK 293 cells. Confocal microscopy of Fluo-3 loaded cells transfected with any of the three RyR2s showed no spontaneous subcellular Ca(2+) release events at baseline. Membrane permeable cAMP analogue (Dioctanoyl-cAMP) triggered subcellular Ca(2+) release events as Ca(2+) sparks and waves. Cells expressing mutant RyR2s showed spontaneous Ca(2+) release events at lower concentrations of cAMP than cells transfected with wild-type RyR2. CONCLUSION: CPVT patients show DADs coinciding with premature action potentials in MAP recordings. Expression studies suggest that DADs are caused by increased propensity of abnormal RyR2s to generate spontaneous Ca(2+) waves in response to cAMP stimulation. Increased sensitivity of mutant RyR2s to cAMP may explain the occurrence of arrhythmias during exercise or emotional stress in CPVT.     

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.     

Ryanodine receptor as a new therapeutic target of heart failure and lethal arrhythmia.

Abnormal intracellular Ca(2+) handling by the sarcoplasmic reticulum (SR) is a critical factor in the development of heart failure (HF). Not only decreased Ca(2+) uptake, but also uncoordinated Ca(2+) release plays a significant role in contractile and relaxation dysfunction. Spontaneous Ca(2+) release through ryanodine receptor (RyR) 2, a huge tetrameric protein, during diastole leads to a decrease in the SR Ca(2+) content, and also triggers delayed after depolarization that is a substrate for lethal arrhythmia. Several disease-linked mutations of RyR have been reported in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) or arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2). The unique distribution of these mutation sites has lead to the concept that an interaction among the putative regulatory domains within RyR may play a key role in regulating channel opening, and that there seems to be a common abnormality in the channel disorder of HF and CPVT/ARVC2. Recent knowledge gained from pathological conditions may lead to the development of a new therapeutic strategy for the treatment of HF or cardiac arrhythmia.     

Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKIIdelta. Here we acutely overexpress CaMKIIdeltaC using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKIIdeltaC transgenic mice. CaMKIIdeltaC protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKIIdeltaC versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (tau(0.5Hz)/tau(3Hz)=2.14+/-0.10 versus 1.87+/-0.10), and peak Ca2+ current (ICa) was increased by 31% (-7.1+/-0.5 versus -5.4+/-0.5 pA/pF, P<0.05). Ca2+ transient amplitude was not significantly reduced (-27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P<0.05). Thus fractional SR Ca2+ release was increased by 60% (P<0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKIIdeltaC versus LacZ myocytes (P<0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKIIdeltaC myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKIIdeltaC overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKIIdeltaC myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.     

Calcium waves driven by "sensitization" wave-fronts

OBJECTIVE: Cellular Ca(2+) waves are understood as reaction-diffusion systems sustained by Ca(2+)-induced Ca(2+) release (CICR) from Ca(2+) stores. Given the recently discovered sensitization of Ca(2+) release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca(2+), waves could also be driven by RyR sensitization, mediated by SR overloading via Ca(2+) pump (SERCA), acting in tandem with CICR. METHODS: Confocal imaging of the Ca(2+) indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. RESULTS: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca(2+) signaling or SR Ca(2+) content due to SERCA inhibition were observed in the short time-frame of these experiments. CONCLUSIONS: Our findings are consistent with Ca(2+) loading resulting in a zone of RyR 'sensitization' traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca(2+) waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca(2+) release.     

A novel mutation in FKBP12.6 binding region of the human cardiac ryanodine receptor gene (R2401H) in a Japanese patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an autosomal dominant inherited disorder characterized by adrenergic induced polymorphic ventricular tachycardias and associated with sudden cardiac death. The human cardiac ryanodine receptor gene (RyR2) was linked to CPVT. A 20-year-old male was referred to our hospital because of recurrent syncope after physical and emotional stress. Routine cardiac examinations including catheterization revealed no structural abnormality. Exercise on treadmill induced premature ventricular contraction in bigeminy and bidirectional ventricular tachycardia was induced during isoproterenol infusion. Beta-blocking drug was effective in suppressing the arrhythmias. We performed genetic screening by PCR-SSCP method followed by DNA sequencing, and a novel missense mutation R2401H in RyR2 located in FKBP12.6 binding region was identified. This mutation was not detected in 190 healthy controls. Since FKBP12.6 plays a critical role in Ca channel gating, the R2401H mutation can be expected to alter Ca-induced Ca release and E-C coupling resulting in CPVT. This is the first report of RyR2 mutation in CPVT patient from Asia including Japan.     

Ca2+/calmodulin kinase II-dependent phosphorylation of ryanodine receptors suppresses Ca2+ sparks and Ca2+ waves in cardiac myocytes

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKIIdelta(C)) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca(2+) release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIdelta(C) via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing beta-galactosidase (beta-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca(2+) transients was slowed, accelerated, or unchanged, whereas spontaneous Ca(2+) spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) groups, respectively. When challenged by high extracellular Ca(2+), both wild-type and constitutively active CaMKIIdelta(C) protected the cells from store overload-induced Ca(2+) release, manifested by a approximately 60% suppression of Ca(2+) waves (at 2 to 20 mmol/L extracellular Ca(2+)) in spite of an elevated sarcoplasmic reticulum Ca(2+) content, whereas dominant-negative CaMKIIdelta(C) promoted Ca(2+) wave production (at 20 mmol/L Ca(2+)) with significantly depleted sarcoplasmic reticulum Ca(2+). Taken together, our data support the notion that CaMKIIdelta(C) negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca(2+) release, thereby affording a negative feedback that stabilizes local and global Ca(2+)-induced Ca(2+) release in the heart.     

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.     

氯沙坦对心力衰竭兔肌浆网钙调控蛋白的影响

目的探讨血管紧张素Ⅱ受体拮抗剂氯沙坦干预慢性心力衰竭对兔心肌肌浆网钙泵（SERCA2）、钙释放通道（RyR2）、受磷蛋白（PLB）基因表达的影响及意义。方法通过结扎兔冠状动脉前降支复制心肌梗死（心梗）模型,以氯沙坦进行干预。于心梗后8周比较观察左室结构、血流动力学的变化及SERCA2、RyR2、PLB基因的表达。结果与对照组相比,心梗组左室舒张末压（LVEDP）显著升高（P〈0．01）,左室压力上升和下降最大速度（＋dr,／dtmax、-dp／dtmax）显著降低（P〈0．01）;氯沙坦组LVEDP显著低于心梗组（P〈0．05）,＋dp／dtmax、-dp／dtmax显著高于心梗组（P〈0．05）。心梗组SERCA2、RyR2、PLBmRNA显著低于对照组（P〈0．01）,而氯沙坦组的上述三项显著高于心梗组（P〈0．05）。结论氯沙坦长期干预心力衰竭,能够改善心脏舒缩功能,可能与其上调肌浆网的钙调控蛋白SERCA2、RyR2、PLB的基因表达有关。     

Suppression of dynamic Ca(2+) transient responses to pacing in ventricular myocytes from mice with genetic calmodulin kinase II inhibition

The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is important for regulating L-type Ca(2+) current (I(Ca)) and cytoplasmic Ca(2+) (Ca(2+)(i)) uptake and release from the sarcoplasmic reticulum (SR), key elements of the 'Ca(2+)-induced Ca(2+) release' (CICR) mechanism. However, the effects of chronic CaMKII inhibition on Ca(2+)(i) responses during CICR are unknown. We hypothesized that chronic CaMKII inhibition significantly affects CICR in ventricular myocytes. We studied CICR by simultaneously measuring Ca(2+)(i) transients and I(Ca) in voltage-clamped ventricular myocytes isolated from a recently developed genetic mouse model of cardiac CaMKII inhibition. These measurements were repeated in ventricular myocytes from novel mice with cardiac CaMKII inhibition lacking phospholamban (PLN), a known CaMKII substrate and a negative regulator of Ca(2+)(i) uptake into the SR Ca(2+) store. CaMKII inhibition eliminated a pattern of I(Ca) increases called facilitation and significantly reduced beat-to-beat and cell-to-cell variability of peak Ca(2+)(i) transients in ventricular myocytes with PLN. PLN ablation eliminated I(Ca) facilitation even in the absence of CaMKII inhibition and the effects of CaMKII inhibition to reduce SR Ca(2+) content and slow SR Ca(2+) uptake were lost in the absence of PLN. PLN ablation significantly reduced I(Ca) beat-to-beat variability in cells with CaMKII inhibition. These findings show that chronic CaMKII inhibition reduces variability of CICR responses in a manner that is partly dependent on the presence of PLN.