Disrupted Junctional Membrane Complexes and Hyperactive Ryanodine Receptors After Acute Junctophilin Knockdown in Mice

Abstract

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 ...

Colchicine Depolymerizes Microtubules, Increases Junctophilin‐2, and Improves Right Ventricular Function in Experimental Pulmonary Arterial Hypertension

“…We propose that increases in JPH2 and improvements in t‐tubule morphology mediate the augmented RV function, which is further supported by the significant relationships between JPH2 and RV function (Figure 5). These results agree with several studies showing that reduced levels of JPH2 are associated with left ventricular dysfunction in animal models36, 37, 38, 39, 40 and human disease states15, 41 and that increased expression of JPH2, achieved via transgenic overexpression,19 antagonism of miR‐24,42 viral‐mediated overexpression,20 or inhibition of calpain proteases,43 improves cardiac function in animal models of LV dysfunction. Thus, increased JPH2 likely mediates the improved RV‐PA coupling in MCT‐colchicine rats.…”

Cardiac BIN1 (cBIN1) is a regulator of cardiac contractile function and an emerging biomarker of heart muscle health

“…Caveonlin-3 organized caveolae distributes along t-tubules and caveolae distribution is altered in Bin1-deleted mouse cardiomyocytes (Laury-Kleintop et al, 2015), indicating crosstalk between cBIN1-microdomains and caveolae microdomains within t-tubules. JPH2 is a t-tubule/jSR junctional protein and is involved in the maintenance of junctional membrane structure, which when knocked down increases the distance between t-tubule and jSR membrane and disrupts jSR membrane complexes (van Oort et al, 2011;Wu et al, 2012;Reynolds et al, 2013). A role of JPH2 in HF progression has been reported (see review in (Takeshima et al, 2015)).…”

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

“…This indirect analysis strategy has been used to detect cell-wide regional changes in TATS component regularity in disease models 7 . For instance, shRNA mediated junctophilin-2 knock-down caused heart failure and the isoform specific protein deficiency resulted in T-tubule reorganization with nanodomain Ca 2+ release dysfunction 8 . We have recently extended the analysis of TATS membrane networks through direct quantitative approaches and further by live cell superresolution microscopy of individual TATS components in mouse VMs using stimulated emission depletion (STED) nanoscopy 9 .…”