Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains

Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (beta AR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopicheart 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 beta AR-cAMP signaling within cardiomyocyte membrane microdomains.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 Forster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and beta AR-cAMP signaling.Results: NAR-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 *p < 0.05). There was no effect of PMU on beta(2)AR-cAMP signaling in RII_Protein Kinase A domains. beta(1)AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that beta(2)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 **p < 0.01, ***p < 0.001 vs. loaded TT). By comparison beta(1)AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (P-o) were reduced by PMU (loaded TT P-o 0.073 +/- 0.011% vs. loaded crest P-o 0.027 +/- 0.006% N,n 5/18-26 mean SEM *p < 0.05) (unloaded TT 0.0350 +/- 0.003% vs. unloaded crest P-o 0.025 N,n 5/20-30 mean SEM NS #p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2.Discussion: PMU suppresses' beta(2)AR-cAMP and LTCC activity. When activated, the signaling of beta(2)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.