Abstract 757: Β-Arrestin-biased Β2-Adrenergic Receptor Signaling Mediates Novel Mechanisms of Cardiomyocyte Contractility

During heart failure, chronically decreased cardiac output can be treated with positive inotropes, but classic inotropes such as β-adrenergic receptor (βAR) agonists that increase cAMP-dependent Ca 2+ mobilization and contractility ultimately enhance patient mortality. Thus, an alternate approach would be to enhance cardiomyocyte contractility without alterations in cAMP and Ca 2+ levels, such as regulation of sarcomeric proteins. Recently, we demonstrated that a small lipidated pepducin designed from the 1 st intracellular loop of β2AR (ICL1-9) enhanced cardiomyocyte contractility in a Ca 2+ -independent, β-arrestin-dependent manner, yet the complete mechanisms remained unclear. We also showed that β2AR stimulation in hearts in vivo or neonatal rat ventricular myocytes (NRVM) in vitro activated RhoA in a βarr-dependent manner. Therefore, we sought to determine both the proximal and distal mechanisms by which ICL1-9 enhances cardiomyocyte contractility. Using adult murine cardiomyocytes isolated from wild-type C57Bl/6J mice, we measured basal, ICL1-9- and isoproterenol (ISO, as a positive control)-promoted contractility either alone or in the presence of inhibitors of Gα i activity (Pertussis toxin), ROCK1 (Y-27632), myosin light chain kinase (ML7), and protein kinase D (CID755673). Inhibition of Gα i activity prior to ICL1-9 stimulation led to a decreased contractile response. Consistent with RhoA activation by ICL1-9, ROCK1 inhibition was able to attenuate ICL1-9-mediated contractility, as was inhibition of MLCK. Interestingly, we observed that inhibition of PKD also attenuated ICL1-9-mediated contractility. These data suggest that ICL1-9 acts proximally to engage a β2AR/Gα i /βarr signaling axis, which may distally increase the activation of kinases including PKD, MLCK, and ROCK to alter the regulation of sarcomeric proteins.