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

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, BCM335, Houston, TX 77030, USA.
Circulation (Impact Factor: 14.43). 02/2011; 123(9):979-88. DOI: 10.1161/CIRCULATIONAHA.110.006437
Source: PubMed


Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ 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(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy.
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 excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance.
Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ 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 Ca2+ release in the heart.

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    • "Muscle from JPH1 KO mice showed that triadic junctions were reduced in number and that the SR was structurally abnormal with vacuolated and swollen structures (Ito et al. 2001; Komazaki et al. 2002). Knockdown of both JPH1 and JPH2 isoforms in cultured adult skeletal muscle fibers leads to deformed triad junctions , impaired SOCE, reduced SR Ca 2? stores and elevated levels of cytosolic Ca 2? concentration (Hirata et al. 2006; van Oort et al. 2011). Ca 2? -dependent proteolysis of JPH1 and JPH2 leads to separation of the membranes and disruption of skeletal excitation–contraction coupling (Murphy et al. 2013). "
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    ABSTRACT: The sarcoplasmic reticulum (SR) of striated muscles is specialized for releasing Ca(2+) following sarcolemma depolarization in order to activate muscle contraction. To this end, the SR forms a network of longitudinal tubules and cisternae that surrounds the myofibrils and, at the same time, participates to the assembly of the triadic junctional membrane complexes formed by the close apposition of one t-tubule, originated from the sarcolemma, and two SR terminal cisternae. Advancements in understanding the molecular basis of the SR structural organization have identified an interaction between sAnk1, a transmembrane protein located on the longitudinal SR (l-SR) tubules, and obscurin, a myofibrillar protein. The direct interaction between these two proteins results in molecular contacts that have the overall effect to stabilize the l-SR tubules along myofibrils in skeletal muscle fibers. Less known is the structural organization of the sites in the SR that are specialized for Ca(2+) release and are positioned at the junctional SR (j-SR), i.e. the region of the terminal cisternae that faces the t-tubule at triads. At the j-SR, several trans-membrane proteins like triadin, junctin, or intra-luminal SR proteins like calsequestrin, are assembled together with the ryanodine receptor, the SR Ca(2+) release channel, into a macromolecular complex specialized in releasing Ca(2+). At triads, the 12 nm-wide gap between the t-tubule and the j-SR allows the ryanodine receptor on the j-SR to be functionally coupled with the voltage-gated L-type calcium channel on the t-tubule in order to allow the transduction of the voltage-induced signal into Ca(2+) release through the ryanodine receptor channels. The muscle-specific junctophilin isoforms (JPH1 and JPH2) are anchored to the j-SR with a trans-membrane segment present at the C-terminus and are capable to bind the sarcolemma with a series of phospholipid-binding motifs localized at the N-terminus. Accordingly, through this dual interaction, JPH1 and JPH2 are responsible for the assembly of the triadic junctional membrane complexes. Recent data indicate that junctophilins seem also to interact with other proteins of the excitation-contraction machinery, suggesting that they may contribute to hold excitation-contraction coupling proteins to the sites where the j-SR is being organized.
    Journal of Muscle Research and Cell Motility 09/2015; DOI:10.1007/s10974-015-9421-5 · 2.09 Impact Factor
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    • "It is currently unclear whether t-tubule disarray during heart failure is pro-arrhythmic. Data from the Wehrens group have indicated that orphaned RyRs exhibit increased activity [186], which might theoretically contribute to greater SR Ca 2+ leak and/or Ca 2+ waves. However, our own data have indicated that Ca 2+ sparks almost exclusively occur at intact dyads in failing cells [6]. "
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    ABSTRACT: Improved treatments for heart failure patients will require the development of novel therapeutic strategies that target basal disease mechanisms. Disrupted cardiomyocyte Ca(2+) homeostasis is recognized as a major contributor to the heart failure phenotype, as it plays a key role in systolic and diastolic dysfunction, arrhythmogenesis, and hypertrophy and apoptosis signaling. In this review, we outline existing knowledge of the involvement of Ca(2+) homeostasis in these deficits, and identify four promising targets for therapeutic intervention: the sarcoplasmic reticulum Ca(2+) ATPase, the Na(+)-Ca(2+) exchanger, the ryanodine receptor, and t-tubule structure. We discuss experimental data indicating the applicability of these targets that has led to recent and ongoing clinical trials, and suggest future therapeutic approaches.
    Current Pharmaceutical Design 12/2014; 21(4):431-48. DOI:10.2174/138161282104141204124129 · 3.45 Impact Factor
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    • "Superresolution analysis of RyR2 and JPH2 showed the relative distributions in the junctional space (Fig. 2B lower right) [5]. Importantly, decreased levels of the JPH2 protein have been shown to destabilize the dyadic CRU architecture and Ca 2+ release [90]. JPH2 is downregulated in heart failure, which we have recently confirmed in a myocardial infarct model [4]. "
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    ABSTRACT: Detailed understanding of the adaptive nature of cardiac cells in health and disease requires investigation of proteins and membranes in their native physiological environment, ideally by noninvasive optical methods. However, conventional light microscopy does not resolve the spatial characteristics of small fluorescently labeled protein or membrane structures in cells. Due to diffraction limiting resolution to half the wavelength of light, adjacent fluorescent molecules spaced at less than ~ 250 nm are not separately visualized. This fundamental problem has lead to a rapidly growing area of research, superresolution fluorescence microscopy, also called nanoscopy. We discuss pioneering applications of superresolution microscopy relevant to the heart, emphasizing different nanoscopy strategies toward new insight in cardiac cell biology. Here, we focus on molecular and structural readouts from subcellular nanodomains and organelles related to Ca2 + signaling during excitation–contraction (EC) coupling, including live cell imaging strategies. Based on existing data and superresolution techniques, we suggest that an important future aim will be subcellular in situ structure–function analysis with nanometric resolving power in organotypic cells. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.
    Journal of Molecular and Cellular Cardiology 05/2013; 58(1):13–21. DOI:10.1016/j.yjmcc.2012.11.016 · 4.66 Impact Factor
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