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.     

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.     

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.     

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.     

FKBP12.6与RyR2的关系及其在心脏疾病中的意义

FKBP12.6从RyR2解离导致通道构象及功能改变。Ca2 从SR漏出使SR的Ca2 负荷减少,Ca2 瞬变减少,舒缩时RyR2耦联障碍,最终引起心肌功能异常。而且RyR2对CICR敏感性提高导致心肌迟后去极诱发心律失常。通过过表达或增加FKBP12.6对RyR2亲和力可以改善通道缺陷,从而使心脏功能及心律失常得到改善。     

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.     

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.     

Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca(2+) sparks

Coupling between L-type Ca(2+) channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca(2+) influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca(2+) release. The L-type Ca(2+) channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca(2+) influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca(2+) spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401+/-15% within 10 seconds in Ca(2+)-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245+/-12% increase in CaSpF. Nifedipine (5 micromol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca(2+) channel agonist FPL-64176 (1 micromol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca(2+) current, I(Ca)). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I(Ca) only slowly (tau=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I(Ca) and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca(2+) channel and RyR.     

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.     

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes

Altered Ca(2+) homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca(2+) homeostasis. Diastolic Ca(2+) levels are elevated in NOS1(-/-) and NOS1/NOS3(-/-) but not NOS3(-/-) myocytes compared with wild-type (WT), suggesting diastolic Ca(2+) leakage. Measured leak was increased in NOS1(-/-) and NOS1/NOS3(-/-) but not in NOS3(-/-) myocytes compared with WT. Importantly, NOS1(-/-) and NOS1/NOS3(-/-) myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1(-/-) hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca(2+) leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.     

Microtubule disruption by colchicine reversibly enhances calcium signaling in intact rat cardiac myocytes

Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (I(Ca)) and [Ca(2+)](i) transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 micromol/L colchicine on Ca(2+) signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca(2+) sparks. Colchicine treatment decreased Ca(2+) spark amplitude (F/F(0): 1.78+/-0.01, n=983, versus 1.64+/-0.01, n=1660, recorded in control versus colchicine-treated cells; P<0.0001) without modifying the sarcoplasmic reticulum Ca(2+) load and enhanced their time to peak (in ms: 6.85+/-0.09, n=1185, versus 7.33+/-0.13, n=1647; P<0.0001). Microtubule disruption also induced the appearance of Ca(2+) sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca(2+) signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca(2+)](i) transient evoked by field stimulation. Cells were loaded with the fluorescence Ca(2+) indicator, Fluo-3 cell permeant, and stimulated at 1 HZ: [Ca(2+)](i) transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca(2+)](i) transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca(2+) signaling in cardiac cells with intact sarcolemma.     

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.     

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.     

Impaired contractile performance of cultured rabbit ventricular myocytes after adenoviral gene transfer of Na(+)-Ca(2+) exchanger

Na(+)-Ca(2+) exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral beta-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min(-1), FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P:<0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027+/-0.002 at 30 min(-1) to 0. 037+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1)) and to 0. 040+/-0.002 at 180 min(-1) (P:<0.05 versus 30 min(-1)). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024+/-0.002 at 30 min(-1), 0.029+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1), P:<0.05 versus control), and 0. 026+/-0.002 at 180 min(-1) (NS versus 30 min(-1), P:<0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca(2+) load, were significantly reduced at 120 min(-1) in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78+/-4% in control and 65+/-3% in the Ad-NCX group (P:<0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.     

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.     

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.     

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.     

K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes

OBJECTIVES: The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. METHODS: The effect of K201 on excitation-contraction (E-C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca(2+) measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. RESULTS: Acute application of <1 micromol/L K201 had no significant effect on E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to 83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content (74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201 completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca(2+) uptake and release. CONCLUSIONS: K201 modifies E-C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca(2+) release during Ca(2+) overload without significantly affecting Ca(2+) transient amplitude.     

Quantitative assessment of the SR Ca2+ leak-load relationship

Increased diastolic SR Ca2+ leak (J(leak)) could depress contractility in heart failure, but there are conflicting reports regarding the J(leak) magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution +/-1 mmol/L tetracaine (to block resting leak). Total cell [Ca2+] does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting [Ca2+]i declined 25% after tetracaine addition (126+/-6 versus 94+/-6 nmol/L; P<0.05). At the same time, SR [Ca2+] ([Ca2+](SRT)) increased 20% (93+/-8 versus 108+/-6 micromol/L). From this Ca2+ shift, we calculate J(leak) to be 12 micromol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. J(leak) also increased nonlinearly with [Ca2+](SRT) with a steeper increase at higher load. We conclude that J(leak) is 4 to 15 micromol/L cytosol per second at physiological [Ca2+](SRT). The data suggest that the leak is steeply [Ca2+](SRT)-dependent, perhaps because of increased [Ca2+]i sensitivity of the ryanodine receptor at higher [Ca2+](SRT). Key factors that determine [Ca2+](SRT) in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).     

Urocortin II enhances contractility in rabbit ventricular myocytes via CRF(2) receptor-mediated stimulation of protein kinase A

OBJECTIVE: Urocortin II (UcnII), a peptide of the corticotropin-releasing factor (CRF) family, exerts profound actions on the cardiovascular system. Direct effects of UcnII on adult cardiomyocytes have not been evaluated before. Our aim was to characterize functional effects of UcnII on cardiomyocytes and to elucidate the underlying signaling pathway(s) and cellular mechanisms. METHODS: Rabbit ventricular cardiomyocytes were stimulated at 0.5 Hz (22-25 degrees C). Unloaded cell shortening (FS, edge detection), [Ca(2+)](i) transients (Fluo-4), and L-type Ca(2+) currents (I(Ca), whole-cell patch clamping) were measured. Sarcoplasmic reticulum (SR) Ca(2+) load was assessed by rapid application of caffeine (20 mmol/L). RESULTS: UcnII increased cell shortening and accelerated relaxation in a time- and concentration-dependent manner (EC(50): 10.7 nmol/L). The inotropic effect of UcnII was maximal at 100 nmol/L (35%+/-11% increase in FS, n=8, P<0.05). The inotropic and lusitropic actions of UcnII were largely eliminated by inhibition of CRF(2) receptors (10 nmol/L antisauvagine-30, n=5) or protein kinase A (PKA, 500 nmol/L H-89, n=5). UcnII increased [Ca(2+)](i) transient amplitude (by 63%+/-35%, n=7, P<0.05) and decreased the time constant for decay (from 800+/-63 to 218+/-27 ms, n=7, P<0.001). UcnII also increased SR Ca(2+) load (by 19%+/-7%, n=7, P<0.05) and fractional Ca(2+) release (from 57%+/-7% to 98%+/-2%, n=7, P<0.01). I(Ca) was augmented by 32.7%+/-10.0% (n=9, P<0.05) and the I(Ca)-V relationship was shifted by -15 mV during UcnII treatment. CONCLUSION: UcnII exerts positive inotropic and lusitropic effects in cardiomyocytes via CRF(2) receptor-mediated stimulation of PKA which augments I(Ca) and SR Ca(2+) load to increase SR Ca(2+) release and [Ca(2+)](i) transients.