*** DUPLICATE COPY WITH ID NO. 38895 ***Anti-apoptotic mechanism of Ursodeoxycholic Acid (UDCA) on hypoxic cardiomyocytes / Noorul Izzati Hanafi

Ursodeoxycholic acid (UDCA), the most hydrophilic bile acid is used as a therapeutic agent in liver related diseases and to eliminate hydrophobic bile acid induced apoptosis in liver. Recently, studies suggested the potential used of UDCA in treating heart related diseases. UDCA is reported cardioprotective against the development of ischemia. However, the mechanism of action in UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to determine the anti-apoptotic mechanisms of UDCA on cardioprotection using an in vitro hypoxic model of neonatal rat cardiomyocytes. Rat heart from newborn (0-2 days old) was isolated for primary cell culture of cardiomyocytes. Hypoxia was induced by using CoCl₂ and hypoxic chamber. Cardiomyocytes were incubated with UDCA (pre-UDCA and post-UDCA) and coincubated with FTY720 (S1P receptor agonist), PTX (Gαᵢ inhibitor). The treated cardiomyocytes were subjected for proliferation assay (MTS assay), beating assessment assay, protein expression (aSMase and nSMase, Hif-1α, caspase-3 and caspase-9, ERK and Akt), ROS generation assay and gene expression (Hif-1α, smpd1, smpd2, caspase-3, caspase-9). The data were analyzed by using sample paired t-test and One-way ANOVA. MTS assay revealed that UDCA was not toxic to cardiomyocytes even at high concentration (250 µm). Results showed that CoCl₂ activates Hif-1α expression, reduces cell viability; reduce beating rate, upregulates nSMase protein, increases ROS production, downregulates ERK and Akt protein expression. Interestingly, pre-UDCA treatment significantly abolished the negative effects of CoCl₂ on cell viability, beating rate, ROS production, ERK and Akt expression in cardiomyocytes. Treatment with PTX partially inhibits the protection of UDCA against CoCl₂ negatives effects on beating rate of cardiomyocytes. Meanwhile, FTY720 showed similarity with UDCA action in downregulating smpd1 gene expression and upregulates caspase-9 gene expression. In conclusion, the current data suggests that UDCA mechanism is mediated partially through Gαᵢ-coupled receptor dependent and independent pathways in protecting cardiomyocytes against hypoxia. This study provides an insight of anti-apoptotic mechanism of UDCA on hypoxic cardiomyocytes.