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.     

Arrhythmogenic mutation-linked defects in ryanodine receptor autoregulation reveal a novel mechanism of Ca2+ release channel dysfunction

Arrhythmogenic cardiac ryanodine receptor (RyR2) mutations are associated with stress-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD). The causative mechanisms of RyR2 Ca2+ release dysregulation are complex and remain controversial. We investigated the functional impact of clinically-severe RyR2 mutations occurring in the central domain, and the C-terminal I domain, a key locus of RyR2 autoregulation, on interdomain interactions and Ca2+ release in living cells. Using high-resolution confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of interaction between fusion proteins corresponding to amino- (N-) and carboxyl- (C-) terminal RyR2 domains, we determined that in resting cells, RyR2 interdomain interaction remained unaltered after introduction of SCD-linked mutations and normal Ca2+ regulation was maintained. In contrast, after channel activation, the abnormal Ca2+ release via mutant RyR2 was intrinsically linked to altered interdomain interaction that was equivalent with all mutations and exhibited threshold characteristics (caffeine >2.5 mmol/L; Ca2+ >150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca2+ handling and interdomain interaction after channel activation that was absent in signals obtained from the central domain mutation. I domain-linked channel instability also occurred in intact RyR2 expressed in CHO cells and in HL-1 cardiomyocytes. These new insights highlight a critical role for mutation-linked defects in channel autoregulation, and may contribute to a molecular explanation for the augmented Ca2+ release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca2+ release channel dysfunction in arrhythmia and SCD.     

Limitations of FKBP12.6-directed treatment strategies for maladaptive cardiac remodeling and heart failure

Sarcoplasmic reticulum (SR) calcium (Ca) leak can be reduced by enhancing FKBP12.6 binding to SR Ca release channels (RyR2) and expression of a “sticky” FKBP12.6^D37S mutant may correct reduced binding stoichiometry in RyR2 from failing hearts. Both calcium/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and protein kinase A (PKA) are activated in heart failure and promote SR Ca leak at RyR2. It is possible that FKBP12.6 dissociation from RyR2 may promote remodeling and that interventions to reassociate FKBP12.6 with RyR2 reflect a future therapeutic strategy. We created transgenic (TG) mice expressing FKBP12.6^D37S and tested their capacity to improve intracellular Ca handling and pathological remodeling in vivo. FKBP12.6^D37S TG mice were cross-bred with CaMKIIδc TG mice, which are known to exhibit pronounced RyR2 dysfunction and heart failure. We observed a significant improvement of post-rest Ca transients and a higher SR Ca content in FKBP12.6^D37S TG mice. In double-TG mice, a marked reduction of SR Ca spark frequency indicated reduced SR Ca leak but neither SR Ca transient amplitude, SR Ca content nor morphological or functional parameters improved in vivo. Likewise, FKBP12.6^D37S TG mice subjected to increased afterload after aortic banding exhibited higher SR Ca load but did not exhibit any improvement in hypertrophic growth or functional decline. Enhancement of FKBP12.6-RyR2 binding markedly reduced RyR2 Ca leak in CaMKIIδc-induced heart failure and in pressure overload. Our data suggest that activation of CaMKIIδc and pressure overload confer significant resistance towards approaches aiming at FKBP12.6-RyR2 reconstitution in heart failure and maladaptive remodeling, although RyR2 Ca leak can be reduced.     

Mechanisms of abnormal calcium homeostasis in mutations responsible for catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia is a heritable arrhythmia unmasked by exertion or stress and is characterized by triggered activity and sudden cardiac death. In this study, we simulated mutations in 2 genes linked to catecholaminergic polymorphic ventricular tachycardia, the first located in calsequestrin (CSQN2) and the second in the ryanodine receptor (RyR2). The aim of the study was to investigate the mechanistic basis for spontaneous Ca2+ release events that lead to delayed afterdepolarizations in affected patients. Sarcoplasmic reticulum (SR) luminal Ca2+ sensing was incorporated into a model of the human ventricular myocyte, and CSQN2 mutations were modeled by simulating disrupted RyR2 luminal Ca2+ sensing. In voltage-clamp mode, the mutant CSQN2 model recapitulated the smaller calcium transients, smaller time to peak calcium transient, and accelerated recovery from inactivation seen in experiments. In current clamp mode, in the presence of beta stimulation, we observed delayed afterdepolarizations, suggesting that accelerated recovery of RyR2 induced by impaired luminal Ca2+ sensing underlies the triggered activity observed in mutant CSQN2-expressing myocytes. In current-clamp mode, in a model of mutant RyR2 that is characterized by reduced FKBP12.6 binding to the RyR2 on beta stimulation, the impaired coupled gating characteristic of these mutations was modeled by reducing cooperativity of RyR2 activation. In current-clamp mode, the mutant RyR2 model exhibited increased diastolic RyR2 open probability that resulted in formation of delayed afterdepolarizations. In conclusion, these minimal order models of mutant CSQN2 and RyR2 provide plausible mechanisms by which defects in RyR2 gating may lead to the cellular triggers for arrhythmia, with implications for the development of targeted therapy.     

Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6)

Dissociation of FKBP12.6 from the cardiac Ca2+-release channel (RyR2) as a consequence of protein kinase A (PKA) hyperphosphorylation of RyR2 at a single amino acid residue, serine-2808, has been proposed as an important mechanism underlying cardiac dysfunction in heart failure. However, the issue of whether PKA phosphorylation of RyR2 can dissociate FKBP12.6 from RyR2 is controversial. To additionally address this issue, we investigated the effect of PKA phosphorylation and mutations at serine-2808 of RyR2 on recombinant or native FKBP12.6-RyR2 interaction. Site-specific antibodies, which recognize the serine-2808 phosphorylated or nonphosphorylated form of RyR2, were used to unambiguously correlate the phosphorylation state of RyR2 at serine-2808 with its ability to bind FKBP12.6. We found that FKBP12.6 can bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2. The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6. Complete phosphorylation at serine-2808 by exogenous PKA disrupted neither the recombinant nor native FKBP12.6-RyR2 complex. Furthermore, binding of site-specific antibodies to the serine-2808 phosphorylation site did not dissociate FKBP12.6 from or prevent FKBP12.6 from binding to RyR2. Taken together, our results do not support the notion that PKA phosphorylation at serine-2808 dissociates FKBP12.6 from RyR2.     

Overexpression of FK-506 binding protein 12.0 modulates excitation contraction coupling in adult rabbit ventricular cardiomyocytes

The effect of the 12-kDa isoform of FK-506-binding protein (FKBP)12.0 on cardiac excitation-contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6+/-0.4 fold, n=5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0-RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca2+ current (I(Ca,L)) or Ca2+ efflux rates via the Na+/Ca2+ exchanger. Ca2+ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca2+ load was approximately 25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of I(Ca,L) to initiate sarcoplasmic reticulum Ca2+ release was observed over a range of values of sarcoplasmic reticulum Ca2+ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca2+. Ca2+ spark morphology was measured in beta-escin-permeabilized cardiomyocytes. Ca2+ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca2+ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca2+ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation-contraction coupling.     

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.     

RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR)

The cardiac ryanodine receptor (RyR2) governs the release of Ca2+ from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca2+ content reaches a critical level, spontaneous Ca2+ release occurs, a process we refer to as store-overload-induced Ca2+ release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca2+ levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca2+ and enhanced the basal level of [3H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca2+ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca2+ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca2+ overload-associated VT.     

Enhanced store overload-induced Ca2+ release and channel sensitivity to luminal Ca2+ activation are common defects of RyR2 mutations linked to ventricular tachycardia and sudden death

Ventricular tachycardia (VT) is the leading cause of sudden death, and the cardiac ryanodine receptor (RyR2) is emerging as an important focus in its pathogenesis. RyR2 mutations have been linked to VT and sudden death, but their precise impacts on channel function remain largely undefined and controversial. We have previously shown that several disease-linked RyR2 mutations in the C-terminal region enhance the sensitivity of the channel to activation by luminal Ca2+. Cells expressing these RyR2 mutants display an increased propensity for spontaneous Ca2+ release under conditions of store Ca2+ overload, a process we referred to as store overload-induced Ca2+ release (SOICR). To determine whether common defects exist in disease-linked RyR2 mutations, we characterized 6 more RyR2 mutations from different regions of the channel. Stable inducible HEK293 cell lines expressing Q4201R and I4867M from the C-terminal region, S2246L and R2474S from the central region, and R176Q(T2504M) and L433P from the N-terminal region were generated. All of these cell lines display an enhanced propensity for SOICR. HL-1 cardiac cells transfected with disease-linked RyR2 mutations also exhibit increased SOICR activity. Single channel analyses reveal that disease-linked RyR2 mutations primarily increase the channel sensitivity to luminal, but not to cytosolic, Ca2+ activation. Moreover, the Ca2+ dependence of [3H]ryanodine binding to RyR2 wild type and mutants is similar. In contrast to previous reports, we found no evidence that disease-linked RyR2 mutations alter the FKBP12.6-RyR2 interaction. Our data indicate that enhanced SOICR activity and luminal Ca2+ activation represent common defects of RyR2 mutations associated with VT and sudden death. A mechanistic model for CPVT/ARVD2 is proposed.     

Catecholaminergic polymorphic ventricular tachycardia: recent mechanistic insights

Cardiac excitation-contraction coupling occurs by a calcium ion-mediated mechanism in which the signal of action potential is converted into Ca2+ influx into the cardiomyocytes through the sarcolemmal L-type calcium channels. This is followed by Ca2+-induced release of additional Ca2+ ions from the lumen of the sarcoplasmic reticulum into the cytosol via type 2 ryanodine receptors (RyR2). RyR2 channels form large complexes with additional regulatory proteins, including FKBP12.6 and calsequestrin 2 (CASQ2). Catecholamines, released into the body fluids during emotional or physical stress, activate Ca2+-induced Ca2+ release by protein kinase A-mediated phosphorylation of RyR2. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an insidious, early-onset and highly malignant, inherited disorder characterized by effort-induced ventricular arrhythmias in the absence of structural alterations of the heart. At least some cases of sudden, unexplained death in young individuals may be ascribed to CPVT. Mutations of the RyR2 gene cause autosomal dominant CPVT, while mutations of the CASQ2 gene may cause an autosomal recessive or dominant form of CPVT. The steps of the molecular pathogenesis of CPVT are not entirely clear, but inappropriate "leakiness" of RyR2 channels is thought to play a role; the underlying mechanisms may involve an increase in the basal activity of the RyR2 channel, alterations in its phosphorylation status, a defective interaction of RyR2 with other molecules or ions, such as FKBP12.6, CASQ2, or Mg2+, or its abnormal activation by extra- or intraluminal Ca2+ ions. Beta-adrenergic antagonists have proven to be of value in prevention of arrhythmias in CPVT patients, but occasional treatment failures call for alternative measures. There is great interest at present for the development of novel antiarrhythmic drugs for CPVT, as the same approaches may be applied for treatment of more common forms of life-threatening arrhythmias, such as those arising during ischemia and heart failure.     

Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death

Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.     

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.     

Abnormal Ca2+ release,but normal ryanodine receptors,in canine and human heart failure

Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44+/-8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30+/-5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P(o) of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor beta-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.     

Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure

Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.     

Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca2+ current. To clarify this, we measured resting Ca2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca2+] was clamped at 50 and 10 nmol/L (and the SR Ca2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca2+ load was changed by the addition of 10 micromol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca2+] increases RyR gating independent of cytosolic [Ca2+] (which was clamped).     

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.     

RyR-bound FKBP12.6 and the modulation

In the pathogenesis of cardiac dysfunction in heart failure, a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+) -ATPase is believed to be a major determinant. Recently, a novel mechanism of cardiac dysfunction in heart failure has been reported on the basis of the following findings:1) PKA hyperphosphorylation of RyR causes a dissociation of FKBP12.6 from RyR, resulting in the abnormal single-channel properties (increased Ca(2+) sensitivity for activation and elevated channel activity associated with destabilization of RyR (Marx et al, Cell 101:365, 2000), 2) a prominent abnormal Ca(2+) leak occurs through RyR, following a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR (Yano M et al, Circulation 102:2131, 2000). This abnormal Ca(2+) leak might possibly cause Ca(2+) overload and consequent diastolic dysfunction, as well as systolic dysfunction.     

Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death

Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca(2+) and an increased propensity for spontaneous Ca(2+) release or store-overload-induced Ca(2+) release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca(2+), but had little effect on the sensitivity of the channel to activation by cytosolic Ca(2+). This specific impact of the A4860G mutation indicates that the luminal Ca(2+) activation of RyR2 is distinct from its cytosolic Ca(2+) activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca(2+) release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca(2+) activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.     

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.