JPH2

Abstract: Rationale: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. Objective: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs. Methods and Results: We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter–driven Cre to Rosa Cas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 ( Jph2 ), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus–transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor. Conclusions: CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Abstract: Background— Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca2+) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca2+ transient amplitudes that were insensitive to L-type Ca2+ channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca2+ levels were observed with potentially increased total Ca2+ stores. Spontaneous Ca2+ oscillations were elicited at a higher extracellular [Ca2+] and with decreased frequency in JPH2 knockdown cells. Conclusions— Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca2+ homeostasis, which may be associated with prohypertrophic remodeling.

Abstract: Aims Chronic heart failure is a complex clinical syndrome with impaired myocardial contractility. In failing cardiomyocytes, decreased signalling efficiency between the L-type Ca2+ channels (LCCs) in the plasma membrane (including transverse tubules, TTs) and the ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) underlies the defective excitation–contraction (E–C) coupling. It is therefore intriguing to know how the LCC–RyR signalling apparatus is remodelled in human heart failure. Methods and results Stereological analysis of transmission electron microscopic images showed that the volume densities and the surface areas of TTs and junctional SRs were both decreased in heart failure specimens of dilated cardiomyopathy (DCM) and ischaemic cardiomyopathy (ICM). The TT–SR junctions were reduced by ∼60%, with the remaining displaced from the Z-line areas. Moreover, the spatial span of individual TT–SR junctions was reduced by ∼17% in both DCM and ICM tissues. In accordance with these remodelling, junctophilin-2 (JP2), a structural protein anchoring SRs to TTs, was down-regulated, and miR-24, a microRNA that suppresses JP2 expression, was up-regulated in both heart failure tissues. Conclusion Human heart failure of distinct causes shared similar physical uncoupling between TTs and SRs, which appeared attributable to the reduced expression of JP2 and increased expression of miR-24. Therapeutic strategy against JP2 down-regulation would be expected to protect patients from cardiac E–C uncoupling.