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 ...

Rationale Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in heart failure (HF) development and associated diastolic sarcoplasmic reticulum (SR) Ca2+ leak is unclear. Objective Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with non-ischemic and ischemic forms of HF. Methods and Results Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with non-ischemic but not with ischemic HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development following transverse aortic constriction (TAC) compared to wildtype (WT) littermates. After TAC, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor (ANF). Cardiomyocytes from S2814A mice exhibited significantly lower SR Ca2+ leak and improved SR Ca2+ loading compared to WT mice after TAC. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice following experimental myocardial infarction. Conclusions Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological SR Ca2+ leak and heart failure development in non-ischemic forms of HF such as transverse aortic constriction in mice.

Objectives To study the role of junctophilin 2 (JPH2) in atrial fibrillation (AF). Background JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca2+ handling and modulation of ryanodine receptor Ca2+ channels (RyR2). Whereas defective RyR2-mediated Ca2+ release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias. Methods Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudo-knockin (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation. Results PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF compared with wildtype (WT)-PKI mice, while A399S-PKI mice expressing a HCM-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca2+ release events. These changes were attributed to reduced binding of E169KJPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca2+ spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients, and an increased frequency of spontaneous Ca2+ release events. Conclusions Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2:RyR2 ratios can promote SR Ca2+ leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.

Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-␣-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.

Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

Junctophilins (JPHs) are members of a junctional membrane complex protein family important for the physical approximation of plasmalemmal and sarcoplasmic/endoplasmic reticulum membranes. As such, JPHs facilitate signal transduction in excitable cells between plasmalemmal voltagegated calcium channels and intracellular calcium release channels. To determine the molecular evolution of the JPH gene family, we performed a phylogenetic analysis of over 60 JPH genes from over 40 species and compared conservation across species and different isoforms. We found that JPHs are evolutionary highly conserved, in particular the membrane occupation and recognition nexus motifs found in all species. Our data suggest that an ancestral form of JPH arose at the latest in a common metazoan ancestor and that in vertebrates four isoforms arose, probably following two rounds of whole genome duplications. By combining multiple prediction techniques with sequence alignments, we also postulate the presence of new important functional regions and candidate sites for posttranslational modifications. The increasing number of available sequences yields significant insight into the molecular evolution of JPHs. Our analysis is consistent with the emerging concept that JPHs serve dual important functions in excitable cells: structural assembly of junctional membrane complexes and regulation of intracellular calcium signaling pathways.

Background-Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca 2ϩ ) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca 2ϩ channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca 2ϩ handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca 2ϩ handling in a prohypertrophic manner. Methods and Results-JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca