Transgenic CaMKII {delta} C overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release (original) (raw)
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Circulation Research, 2005
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 ph...
Circulation Research, 2006
The predominant cardiac Ca 2ϩ /calmodulin-dependent protein kinase (CaMK) is CaMKII␦. Here we acutely overexpress CaMKII␦ C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII␦ C transgenic mice. CaMKII␦ C protein expression and activation state (autophosphorylation) were increased 5-to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII␦ C versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced ( 0.5Hz / 3Hz ϭ2.14Ϯ0.10 versus 1.87Ϯ0.10), and peak Ca 2ϩ current (I Ca ) was increased by 31% (Ϫ7.1Ϯ0.5 versus Ϫ5.4Ϯ0.5 pA/pF, PϽ0.05). Ca 2ϩ transient amplitude was not significantly reduced (Ϫ27%, Pϭ0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca 2ϩ content (41%; PϽ0.05). Thus fractional SR Ca 2ϩ release was increased by 60% (PϽ0.05). Diastolic SR Ca 2ϩ leak assessed by Ca 2ϩ spark frequency (normalized to SR Ca 2ϩ load) was increased by 88% in CaMKII␦ C versus LacZ myocytes (PϽ0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca 2ϩ leak may explain reduced SR Ca 2ϩ content, despite measured levels of SR Ca 2ϩ -ATPase and Na ϩ /Ca 2ϩ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKII␦ C myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKII␦ C overexpression alters RyR function, leading to enhanced SR Ca 2ϩ leak and reduced SR Ca 2ϩ content but without reducing twitch contraction and Ca 2ϩ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca 2ϩ release in CaMKII␦ C myocytes (ie, CaMKII-dependent enhancement of RyR Ca 2ϩ sensitivity during diastole and systole) and increased I Ca . (Circ Res. 2006;98:235-244.)
Circulation, 2010
Background Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca 2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2+ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results Mice in which the S2814 Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2+ release events, resulting in reduced sarcoplasmic reticulum Ca 2+ load on confocal microscopy. These Ca 2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts wi...
Basic Research in Cardiology
Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibiti...
PloS one, 2014
In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mic...
Circulation, 2013
Background— Sarcoplasmic reticulum (SR) Ca 2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca 2+ leak. In HF, however, the SR Ca 2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca 2+ transients, a depletion of SR Ca 2+ storage and elevated diastolic Ca 2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA...
Abnormal Ca2+ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure
Circulation Research, 2002
Sarcoplasmic reticulum (SR) Ca 2ϩ 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 Ca 2ϩ transport. Intracellular Ca 2ϩ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca 2ϩ ] i transient were reduced in HF. Ca 2ϩ uptake assays showed 44Ϯ8% reduction of V max in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30Ϯ5% 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 -adrenergic stimulation of Ca 2ϩ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca 2ϩ uptake, more than Ca 2ϩ release, contributing to the depressed and slow Ca 2ϩ transient characteristic of HF. (Circ Res. 2002;91:1015-1022.)
Circulation Research, 2010
Rationale : We previously showed that transgenic mice expressing Ca 2+ /calmodulin-dependent protein kinase II δ C (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca 2+ leak and lowering of SR Ca 2+ load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca 2+ load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG. Objective : Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIδ C expression were corrected, and establish the mechanistic basis for these changes. Methods and Results : CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca 2+ load and twitch Ca 2+ transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart fai...