Abstract We122: Suppression of defender against cell death 1 (Dad1) induces cardiomyocyte death by regulating cell adhesion proteins.

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Introduction: Suppression of cardiomyocyte loss is considered a promising strategy for the prevention of the onset of heart failure (HF). Previously, defender against cell death 1 ( Dad1 ) was identified as a cytoprotective gene. Dad1 forms a complex with staurosporine and temperature sensitive 3A (Stt3A), a catalytic subunit of oligosaccharyltransferase (OST) complex which is responsible for N-glycosylation. However, the function of Dad1 in cardiomyocytes remains unknown. Hypothesis: The disruption of cell-extracellular matrix interactions results in cell death, known as anoikis. Because various cell adhesion proteins are N-glycosylated, we hypothesized that Dad1 suppressed anoikis by regulating N-glycosylation of cell adhesion proteins in cardiomyocytes. Methods: Neonatal rat cardiomyocytes were cultured and the expression of Dad1 or Stt3A was knocked down using siRNA. The protein expression was assessed with immunoblot analysis, and cell viability was evaluated using a CellTiter-Blue assay kit. Results: The suppression of Dad1 using siRNAs reduced cardiomyocyte viability ( p <0.01, n=4). Interestingly, suppression of Dad1 impaired the N-glycosylation of integrin β1 and decreased the expression of mature integrin β1 ( p <0.01, n=3). These changes inactivated integrin-mediated signal transduction through dephosphorylation of focal adhesion kinase ( p <0.01, n=5). In addition, enhanced cell-extracellular matrix interactions by using adhesamine rescued the cardiomyocyte death by Dad1 knockdown ( p <0.01, n=4). Next, we pursued the relationship between Dad1 and Stt3A. Dad1 knockdown decreased the expression of Stt3A ( p <0.01, n=6). Suppression of Stt3A induced cardiomyocyte death accompanied by the changes of cell adhesion proteins ( p <0.01, n=4). Finally, double knockdown of Dad1 and Stt3A did not induce a further reduction in cell viability compared with the knockdown of only Dad1 ( p <0.05, n=4), suggesting that cardiomyocyte death by Dad1 knockdown was dependent on Stt3A. Conclusion: Dad1 is required for the stabilization of Stt3A and suppresses cardiomyocyte anoikis by regulating N-glycosylation of cell adhesion proteins in an OST-dependent manner. Dad1-mediated cardiomyocyte survival could be a therapeutic target against HF.