Background In a canine model of premature ventricular contraction-induced cardiomyopathy (PVC-CM), Cav1.2 is downregulated and misplaced from t-tubules. Junctophilin-2 (JPH-2) is also downregulated. Objective 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 ultra-structures (electron microscopy), probe JPH-2 interacting proteins (co-immunoprecipitation), quantify dyad and non-dyad 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 ultra-structures. Among nineteen 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 of dyad protein levels when JPH-2 is downregulated. Furthermore, several dyad proteins, including Na/Ca exchanger, are missing or shifted from dyads to 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 ICaL (Cav1.2/Cavβ2) and IKs (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.
Background Frequent premature ventricular contractions (PVC) have been associated with PVC-induced cardiomyopathy (CM) in some patients. Objective Understand the cardiac consequences of different PVC burden and minimum burden required to induce LV dysfunction. Methods RV apical PVCs at a coupling interval of 240ms were introduced at different PVC burden in 9 mongrel canines. A stepwise increase in PVC burden was implemented every 8 weeks from 0% (baseline), 7%, 14%, 25%, 33% to 50% using our premature pacing algorithm. Echocardiogram and 24-hr Holter were obtained at 4- and 8-week period at each PVC burden with a single blinded reader assessing all echocardiographic parameters including speckle tracking imaging (GE EchoPAC). CM was defined as LVEF < 50% or a decrease >10% points. IL-6 and pro-BNP levels were obtained at the end of each PVC burden. Results LVEF±SD (mean heart rate ± SD) for 0%, 7%, 14%, 33%, and 50% at 8 weeks for each PVC burden were 57±2.9% (85±13bpm); 54.4±3% (81±10bpm); 53.3±5% (77±12bpm); 51.1±4.2% (79±14bpm); 47.7±3.8% (80±14bpm); 44.7±1.9% (157±43bpm). PVC-induced CM was present in 11.1%, 44.4%, and 100% of animals with 25%, 33% and 50% PVC burden, respectively. E/A ratio and radial strain decreased while LA size increased beyond 33% PVC burden. No changes in pro-BNP and IL-6 levels were noted at any PVC burden. Conclusion LV systolic function (LVEF, and radial strain) declined linearly as PVC burden increased. PVC-induced CM developed in some canines with 25% and 33% PVC burden, but developed in all animals with 50% PVC burden.
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