THE EMERGING ROLE OF THE INTERACTION BETWEEN JUNCTOPHILIN-2 AND L-TYPE CALCIUM CHANNEL IN EXCITATION-CONTRACTION COUPLING MICRO-SIGNALING DOMAINS DURING CARDIAC PATHOLOGICAL REMODELING

Abstract

Pathological cardiac remodeling is a set of cellular and molecular changes culminating in ventricular dysfunction, malignant arrhythmias and heart failure. Prominent effects of pathological cardiac remodeling include loss of transverse tubules (T-tubules) and disruption of cardiac dyads. The dyad composes the basic microstructural element in cardiomyocytes by forming a junctional complex, in which the T-tubular membrane and the junctional sarcoplasmic reticulum (jSR) membrane are brought into a close proximity. In this spatially restricted microdomain, the L-type Ca2+ channel (LTCC) in the T-tubules is closely located to the juxtaposed Ryanodine receptors (RyRs) in the jSR membrane. Ca2+ influx through LTCC triggers Ca2+ release from the RyRs, in a process known as Ca2+ induced Ca2+ release (CICR), which enables excitation-contraction coupling (EC coupling). Under physiological conditions, the majority of LTCCs reside in the T-tubules. However, upon disruption of the cardiac dyad complexes during pathological remodeling, LTCCs are redistributed away from the T-tubules, leading to defective EC coupling and abnormal CICR. The molecular mechanism responsible for LTCCs recruitment to the T-tubules, or their redistribution away from the T-tubules under pathological remodeling in cardiac diseases, is not fully elucidated. Junctophilin-2 (JPH2) is a crucial regulator of the dyad structure that provides a structural bridge of 12-15nm between the plasma membrane (PM) in the T-tubules and the jSR. By stabilizing the dyad structure, JPH2 enables the functional crosstalk between LTCCs and RyRs to ensure a proper CICR. While most of the JPH2 domains functions are well known, the role of the ‘Joining region’, which is located between two PM interacting domains in JPH2, remains unknown. Moreover, it remains unexplored if the Joining region in JPH2 directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules. The overarching theme of this dissertation is to determine the role of the Joining region in JPH2 in cardiomyocytes and to explore if the Joining region in JPH2 recruits LTCC to T-tubules via direct interaction that promotes order to enable efficient CICR. We validated that pathological remodeling in in vivo feline model with progressive pressure overload involves alterations of JPH2 abundance and LTCC redistribution across the cardiomyocyte PM. Similar changes with JPH2 and LTCC expressions were observed in in vitro models of cultured adult feline ventricular myocytes (AFVMs). Adenovirus-mediated overexpression of mutated JPH2 in the Joining region (mutPG1JPH2) in AFVMs induced severe T-tubules remodeling and dyad degradation. Protein-protein interaction studies showed that the Joining region in JPH2 interacts with the pore-forming subunit α1C in LTCC. In addition, our data showed that JPH2 elicits LTCC distribution to dyads, where it colocalizes with the Ryanodine receptor. The interaction between LTCC and JPH2 was crucial for T-tubule stabilization. Disruption of this interaction introduced asynchronous Ca2+ release with impaired EC coupling that could be detected after β-adrenergic stimulation. Overall, Ca2+ imbalance in mutPG1JPH2 overexpressing AFVMs induced Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation and altered the myocyte bioenergetics. Collectively, the data presented in this dissertation provides extensive evidence that the interaction between LTCC and the Joining region in JPH2 facilitates dyad assembly and regulates appropriate CIRC in cardiomyocytes.