JPH-2 interacts with Cai -handling proteins and ion channels in dyads: Contribution to premature ventricular contraction–induced cardiomyopathy

• Published in: Heart rhythm Vol. 13; no. 3; pp. 743 - 752
• Main Authors: Jiang, Min, PhD, Zhang, Mei, PhD, Howren, Maureen, BS, Wang, Yuhong, PhD, Tan, Alex, MD, Balijepalli, Ravi C., PhD, Huizar, Jose F., MD, FHRS, FAHA, Tseng, Gea-Ny, PhD, FAHA
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2016
• Subjects: Animals; Cardiomyopathies - etiology; Cardiomyopathies - metabolism; Cardiomyopathies - pathology; Cardiomyopathy; Cardiovascular; Cells, Cultured; Disease Models, Animal; Dogs; Dyad; Excitation-contraction coupling; Immunoblotting; Membrane Proteins - biosynthesis; Microscopy, Confocal; Microscopy, Electron; Myocardium - metabolism; Myocardium - pathology; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - ultrastructure; Premature ventricular contractions; Sarcoplasmic reticulum; Sodium-Calcium Exchanger - biosynthesis; T tubules; Ventricular Premature Complexes - complications; Ventricular Premature Complexes - metabolism; Ventricular Premature Complexes - pathology; Ab; ryanodine receptor 2; NCX; Cardiomyopathy; jSR; Ca-induced Ca release; CICR; LV; sarcoplasmic reticulum; BIN-1; Super; voltage-gated Ca channel β subunit 2; T tubules; slow delayed rectifier current; Na/Ca exchanger; left ventricular; wheat germ agglutinin; I Ks; L-type Ca channel current; JPH-2; SR; RyR2; T tubule; antibody; CON; I CaL; bridging integrator-1; IP; control; PVC myocytes; PVC hearts; junctophilin-2; CSQ2; WGA; Premature ventricular contractions; supernatant after immunoprecipitation; Excitation-contraction coupling; immunoprecipitate; calsequestrin 2; transverse tubule; myocytes isolated from PVC hearts; Dyad; hearts exposed to premature ventricular contraction (PVC); Cav β 2; junctional sarcoplasmic reticulum; ICaL; IKs; Sarcoplasmic reticulum; Cavβ2
• ISSN: 1547-5271; 1556-3871
• DOI: 10.1016/j.hrthm.2015.10.037

Background In a canine model of premature ventricular contraction–induced cardiomyopathy (PVC-CM), Cav1.2 is downregulated and misplaced from transverse tubules (T tubules). Junctophilin-2 (JPH-2) is also downregulated. Objectives The objectives of this study were to understand the role of JPH-2 in PVC-CM and to probe changes in other proteins involved in dyad structure and function. Methods We quantify T-tubule contents (di-8-ANEPPS fluorescence in live myocytes), examine myocyte ultrastructures (electron microscopy), probe JPH-2–interacting proteins (co-immunoprecipitation), quantify dyad and nondyad protein levels (immunoblotting), and examine subcellular distributions of dyad proteins (immunofluorescence/confocal microscopy). We also test direct JPH-2 modulation of channel function (vs indirect modulation through dyad formation) using heterologous expression. Results PVC myocytes have reduced T-tubule contents but otherwise normal ultrastructures. Among 19 proteins examined, only JPH-2, bridging integrator-1 (BIN-1), and Cav1.2 are highly downregulated in PVC hearts. However, statistical analysis indicates a general reduction in dyad protein levels when JPH-2 is downregulated. Furthermore, several dyad proteins, including Na/Ca exchanger, are missing or shifted from dyads to the peripheral surface in PVC myocytes. JPH-2 directly or indirectly interacts with Cai -handling proteins, Cav1.2 and KCNQ1, although not BIN-1 or other scaffolding proteins tested. Expression in mammalian cells that do not have dyads confirms direct JPH-2 modulation of the L-type Ca channel current (Cav1.2/voltage-gated Ca channel β subunit 2) and slow delayed rectifier current (KCNQ1/KCNE1). Conclusion JPH-2 is more than a “dyad glue”: it can modulate Cai handling and ion channel function in the dyad region. Downregulation of JPH-2, BIN-1, and Cav1.2 plays a deterministic role in PVC-CM. Dissecting the hierarchical relationship among the three is necessary for the design of therapeutic interventions to prevent the progression of PVC-CM.