ABSTRACT. Ursodeoxycholic acid (UDCA) is used to treat liver diseases and demonstrates cardioprotective effects. Accumulation of the plasma membrane sphingolipid sphingomyelin in the heart can lead to atherosclerosis and coronary artery disease. Sphingomyelinases (SMases) break down sphingomyelin, producing ceramide, and inhibition of SMases activity can promote cell survival. We hypothesized that UDCA regulates activation of ERK and Akt survival signaling pathways and SMases in protecting cardiac cells against hypoxia. Neonatal cardiomyocytes were isolated from 0-to 2-day-old Sprague Dawley rats, and given 100 µM CoCl 2 , 150 µM H 2 O 2 , or placed in a hypoxia chamber for 24 h. The ameliorative effects of 100-µM UDCA treatment for 12 h were then assessed using MTS, QuantiGene Plex (for Smpd1 and Smpd2), and SMase assays, beating rate assessment, and western blotting (for ERK and Akt). Data were analyzed by the paired Student t-tests and one-way analyses of variance. Cell viability decreased significantly after H 2 O 2 (85%), CoCl 2 (50%), and hypoxia chamber (52%) treatments compared to the untreated control (100%). UDCA significantly counteracted the effects of chamber-and CoCl 2 -induced hypoxia on viability and beating rate. However, no significant differences were observed in acid SMase gene and protein expression between the untreated, CoCl 2 , and UDCA-CoCl 2 groups. In contrast, neutral SMase gene and protein expression did significantly differ between the latter two groups. ERK and Akt phosphorylation was higher in hypoxic cardiomyocytes treated with UDCA than those given CoCl 2 alone. In conclusion, UDCA regulates the activation of survival signaling proteins and SMases in neonatal rat cardiomyocytes during hypoxia.
Ursodeoxycholic acid (UDCA) is known as a therapeutic agent in treating cholestasis and liver diseases. Recently, UDCA has been suggested as a therapeutic drug for heart related diseases. Cardioprotective effect of UDCA against the development of ischemia has been studied. Yet, the mechanism of UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to elucidate the mechanisms of UDCA cardioprotection against hypoxia by investigating the expression of caspase -3/-9 and ROS generation using an in vitro hypoxic heart model. A newborn (0-2 days old) rat heart was isolated for primary cell culture of cardiomyocytes. Hypoxia was chemically induced by using CoCl2. Cardiomyocytes were then incubated with UDCA. The treated cardiomyocytes were subjected for ROS generation detection assay, QuantiGene Plex assay for caspase-3/-9 gene expression and ELISA for caspase-3/-9 protein expression. The data were analyzed by using sample paired t-test and One-way ANOVA. Our results showed that UDCA abolishes the effects on CoCl2 in ROS production and UDCA downregulates caspase-9 protein expression in CoCl2 treated cardiomyocytes. This study provides an insight of UDCA in protecting cardiomyocytes against hypoxia mediated by anti-apoptosis mechanism.
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