Image_1_Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains.tif WrightPeter T. Sanchez-AlonsoJose L. LucarelliCarla Alvarez-LaviadaAnita PouletClaire E. BelloSean O. FaggianGiuseppe M. TerraccianoCesare GorelikJulia 2018 <p>Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains.</p><p>Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling.</p><p>Results: β<sub>2</sub>AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM <sup>∗</sup>p < 0.05). There was no effect of PMU on β<sub>2</sub>AR-cAMP signaling in RII_Protein Kinase A domains. β<sub>1</sub>AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β<sub>2</sub>AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM <sup>∗∗</sup>p < 0.01, <sup>∗∗∗</sup>p < 0.001 vs. loaded TT). By comparison β<sub>1</sub>AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (P<sub>o</sub>) were reduced by PMU (loaded TT P<sub>o</sub> 0.073 ± 0.011% vs. loaded crest P<sub>o</sub> 0.027 ± 0.006% N,n 5/18-26 mean ± SEM <sup>∗</sup>p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest P<sub>o</sub> 0.025 N,n 5/20-30 mean ± SEM NS <sup>#</sup>p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2.</p><p>Discussion: PMU suppresses’ β<sub>2</sub>AR-cAMP and LTCC activity. When activated, the signaling of β<sub>2</sub>AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of ‘suppression/decompartmentation’ is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.</p>