Abstract 20956: The Role of Junctophilin 2 in Calcium Release Unit Formation in Cardiomyocytes
Introduction and Hypothesis: Efficiency of excitation contraction coupling in cardiomyocytes is dependent on the distance between the plasma membrane (PM) and the SR membrane (SRM). Optimal distance ensures formation of calcium release units (CRU) by coupling voltage gated calcium channel (VGCC) on PM and ryanodine receptor-2 (RyR2) on SRM, respectively. Close approximation of VGCC and RyR2 within CRUs is believed to be mediated by junctophilin 2 (JPH2). Recently loss-of-function mutations in JPH2 were found in patients with hypertrophic cardiomyopathy. We hypothesize that in cardiomyocytes, JPH2 plays a role in ECC by contributing to the formation of CRUs through its physical interactions with RyR2 and the other proteins.
Methods and Results: We generated inducible, cardiac-specific JPH2 knockdown mice using RNA interference of JPH2 (JPH2 KD). JPH2 KD mice display an increased mortality with enlarged cardiac dimensions (heart to body weight = 7.3±0.4 vs. 5.8±0.2 mg/g, KD vs. control, p<0.001; end-diastolic dimension by transthoracic echocardiography = 4.73±0.06 vs. 4.39±0.06 mm, KD vs. control, p<0.001). At subcellular level, electron microscopy data showed a high variance in the distance between PM and SRM (3.22 vs. 1.66 nm2, KD vs. control, p=0.005), and a decrease in number of sarcomere-neighboring CRUs in the KD cardiomyocytes. Inspite of this disarray of CRUs and a decrease in JPH2 protein to 50–60% of control level, levels of RyR2 and VGCC are unaltered in KD mice. Therefore, effect of JPH2 KD on the spatial relationship between RyR2 and VGCC was measured using immunofluorescent confocal imaging. Relative co-localization of the two channels is decreased in KD mice ((coefficient of determination)2 = 0.15±0.02 vs. 0.30±0.04, KD vs. control; p=0.003). Interestingly, RyR2 and the remaining JPH2 continue to co-localize in KD mice, implying criticality of the interaction for CRU formation. Our co-immunoprecipitation assays in wild type heart lysates confirmed direct association between JPH2 and RyR2.
Conclusion: JPH2 directly interacts with RyR2 and is critical for CRU formation in cardiomyocytes. Knockdown of JPH2 disrupts the number and structure of CRUs, which in turn might contribute to cardiac remodeling in JPH2 KD mice.
- © 2010 by American Heart Association, Inc.