Background-Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2ϩ channels and Ca 2ϩ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca 2ϩ -induced Ca 2ϩ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results-To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitationcontraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance. Conclusions-Loss of JPH2 had profound effects on Ca 2ϩ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2ϩ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca 2ϩ release in the heart. (Circulation. 2011;123:979-988.)Key Words: calcium Ⅲ excitation Ⅲ heart failure Ⅲ junctophilin Ⅲ sarcoplasmic reticulum E xcitation-contraction (EC) coupling is the fundamental mechanism by which depolarization of the voltage-gated Ca 2ϩ channels (VGCCs) in the plasmalemma triggers a much greater release of Ca 2ϩ from the sarcoplasmic reticulum (SR) via type 2 ryanodine receptors (RyR2), a process known as Ca 2ϩ -induced Ca 2ϩ release (CICR). 1 This Ca 2ϩ release amplification depends on the organization of VGCC and RyR2 within junctional membrane complexes (JMCs), also known as calcium release units. 2 Disruption of JMC structure, as seen in heart failure, profoundly affects CICR and thus cardiac muscle contractility. 3 Clinical Perspective on p 988The molecular mechanisms involved in organizing Ca 2ϩ channels within the JMC remain poorly understood. One family of proteins, known as junctophilins (JPHs), has been proposed to provide a structural bridge between the plasmalemma and SR, thereby potentially ensuring approximation of VGCC and RyR2. 4 Junctophilin-2 (JPH2) is the major cardiac isoform among the 4 JPH isoforms, which are expressed within JMCs of all excitable cell types. 5 JPH proteins comprise 8 N-terminal "membrane occupation and recognition nexus" domains, a space-spanning ␣-helix, and a C-terminal transmembrane domain. The membrane occupation and recognition nexus domains mediate binding to the plasmalemma, and the hydrophobic transmembrane domain is anchored into ...

Background-Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias.Although abnormal Ca 2ϩ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2ϩ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2ϩ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results-Mice in which the S2814 Ca 2ϩ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2ϩ release events, resulting in reduced sarcoplasmic reticulum Ca 2ϩ load on confocal microscopy. These Ca 2ϩ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca 2ϩ /calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions-Our results suggest that Ca 2ϩ /calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca 2ϩ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure. (Circulation. 2010;122:2669-2679.)Key Words: arrhythmia Ⅲ calcium Ⅲ calcium-calmodulin-dependent protein kinase type 2 Ⅲ heart failure Ⅲ ryanodine receptor calcium release channel Ⅲ sarcoplasmic reticulum C ongestive heart failure (HF) is a leading cause of mortality and morbidity worldwide. Approximately 50% of HF patients die of sudden cardiac death (SCD) attributed to ventricular arrhythmias (Ͼ300 000 in the United States annually). 1,2 A large fraction of these arrhythmias are thought to be initiated by focal triggered mechanisms, such as spontaneous diastolic Ca 2ϩ release from cardiac myocyte ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR), which activates an arrhythmogenic depolarizing inward Na ϩ /Ca 2ϩ exchange (NCX) current. 3,4 Indeed, in HF there is enhanced diastolic SR Ca 2ϩ release and other changes in electrophysiological substrate that greatly enhance the propensity for triggered arrhythmias. Likewise, patients with inherited RyR2 point mutations exhibit catecholaminergic polymorphic ventricular tachycardia, a known cause of SCD with sensitivity to adrenergic conditions such as exercise or stress. 5,6 HF is a chronic hyperadrenergic state, and a prominent theory suggested that ␤-adrenergic activation of protein kinase A (PKA) destabilized ...