P2-54 Role of adrenergic receptor singnaling in embryonic ventricular cell proliferation and differentiation Brittney Allen, Kishore Pasumarthi Dalhousie University, Halifax, NS, Canada In adult hearts, cardiomyocytes (CM) that die in response to aging or pathological insults are replaced by scar tissue. Transplan- tation of embryonic cardiac progenitor cells (CPC) was shown to increase the contractile function of a failing heart. We recently demonstrated that a non-selective beta-adrenergic receptor (β-AR) agonist isoproterenol (ISO) can decrease proliferation of CPCs in vitro and reduce graft size after intracardiac cell transplantation. Whereas, a β 1 -AR antagonist (Metoprolol) abrogated the anti-proliferative effects of ISO and increased graft size. Carvedilol, a commonly used heart failure medication is known to block both α- and β-AR subtypes. There is no information available on the expression profiles of different α-AR subtypes during cardiac ontogeny and whether these receptors play any role in proliferation and differentiation of embryonic ventricular cells. It is hypothesized that expression of alpha 1 AR subtypes are differentially regulated during embryonic heart development and alpha 1 AR signaling plays an important role in ventricular cell proliferation and differentiation. Total RNA samples isolated from different developmental stages of embryonic ventricles were processed for quantitative RT-PCR analysis using α1-AR subtype specific primers. These experiments revealed that α1B or α1D gene expression levels were significantly higher than that of α1A at several stages of cardiac development. Subcellular localization of alpha 1 AR subtypes in embryonic ventricular cells revealed the presence of alpha 1 A, B, and D subtypes in the nucleus as well as the cytoplasm. Embryonic ventricular cultures treated with Carvedilol in the presence or absence of ISO did not show any changes in cell size compared to control cultures. Additionally, cells treated with Carvedilol and Prazosin, resulted in no change in proliferation and differentiation status of CPC and CM cells. Therefore, these results suggest that it may be safe to use non-selective adrenergic receptor blockers with cell transplantation studies. doi:10.1016/j.yjmcc.2018.07.116 P2-55 Epigenetic regulation of cardiac fibroblast senescence by class I histone deacetylases and ING2 Marina Feslibino, Timothy McKinsey University of Colorado, Denver, USA Fibrosis is estimated to contribute to 45% of deaths in the western world. A key cellular mediator of fibrosis is the activated myofibroblast, which is characterized by the secretion of extracellu- lar matrix (ECM) proteins. Myofibroblasts are thought to initially have a positive mechanical influence following an injury. However, long-term activation of these cells can lead to excessive ECM accumulation, culminating in fibrosis and organ failure. Cellular senescence is an irreversible form of cell cycle arrest, associated with a senescence-associated secretory phenotype (SASP), which is characterized by production of ECM-degrading enzymes such as matrix metalloproteases. Thus, it has been proposed that induction of the SASP might be a promising strategy for the resolution of organ fibrosis. In this context, histone deacetylase (HDAC) inhibitors might be candidate therapies for senescence modulation, as this class of enzymes is intensely involved in the control of cellular proliferation. Here, adult rat ventricular fibroblasts stimulated with TGF-β were used as a cell-based model of myofibroblast activation, and a panel of isoform-selective HDAC inhibitors was tested. Inhibitors of class I HDACs (especially HDAC1 and HDAC2), but not class IIa or IIb HDACs, potently stimulated cardiac fibroblast senescence, as assessed by upregulation of p16 and p21 gene expression, enhanced b-galactosidase activity, and induction of the classical SASP pathway. The mechanism of stimulation of senescence by class I HDAC inhibitors appears to involve ING2, a methyl-histone reader protein and phosphoinositide receptor. These findings define a chromatin signaling program for cardiac fibroblast senescence that could be could targeted with ‘epigenetic therapies’ such as small molecule HDAC inhibitors to treat fibrosis of the heart. doi:10.1016/j.yjmcc.2018.07.117 P2-56 Branched chain α-ketoacids: Novel Regulator of Insulin and mTOR Signalling in Skeletal and Cardiac Muscle Dipsikha Biswas a , Andrew Cowie a , Daniel Kane b , Mohamed Touiaibia c , Petra Kienesberger a , Thomas Pulinilkunnil a a Dalhousie Medicine New Brunswick, Saint John, New Brunswick, Canada b St. Francis Xavier University, Antigonish, Nova Scotia, Canada c Université de Moncton, Moncton, New Brunswick, Canada Background: Branched chain α-keto acids (BCKA) are intracellular catabolic product of branched-chain amino acids (BCAAs) catabolism. Mitochondrial oxidation of BCKA is catalyzed by branched chain ketoacid dehydrogenase (BCKDH), enzyme sensitive to inhibitory phosphorylation by branched chain ketoacid dehydrogenase kinase (BCKDK). Skeletal and cardiac muscle generate significant BCKA. Defective BCAA catabolism and elevated BCKA is central to the pathogenesis of obesity, insulin resistance and heart disease. However, the effect of BCKA on muscle insulin signalling is unexplored. Methods and Results: To examine the direct effect of BCKAs on muscle insulin signaling, adult and neonatal rat cardiomyocytes (ARMCs & NRCM), mouse (C2C12) and rat (L6) skeletal myocyte were incubated with 0.4mM palmitate for 16 h followed by 30 mins 5mM BCKA (ketoleucine or ketovaline or ketoisoleucine) and 15 min 100 nM insulin stimulation. Immunoblot analysis revealed that in the absence and presence of palmitate, ketoleucine, ketovaline and to a lesser extent ketoisoleucine significantly reduced insulin-stimulated AKT Serine 473 and 308 phosphorylation with concomitant upreg- ulation of mTOR signaling pathway. Acute exposure of ARCM to ketoleucine alone augmented electron transport chain capacity given elevated complex III/IV respiratory rate for TMPD/ascorbate after treatment with FCCP suggesting that BCKA directly impairs insulin signaling and remodels mitochondrial respiration. To simulate altered intramyocellular BCKA flux, BCKDH and BCKDK were adenovirally modified. Genetic (adenoviral) and pharmacological activation of BCKDH using BCKDK inhibitor BT-2 in C2C12 cells potentiated insulin signaling with a corresponding decline in the phosphorylation of ribosomal p70S6K indicating reduction in mTOR signaling. Overexpression of BCKDK diminished insulin signaling with concomitant hyperactivation of the mTOR signaling. Conclusion: BCKDH and BCKDK exerts control of intramyocellular BCKA flux into mitochondrial oxidation thereby dictating BCKA Abstracts 123