Evaluation of the mechanism of apoptosis in cardiac myocyte with or without sympathetic innervation by Anthracycline

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To clarify whether sympathetic innervation protects the apoptosis by Anthracyclines in rat cardiac myocyte, we cultured 1 day old neonatal rat cardiac myocytes which was not innervated until 1 day old, and co-cultured cardiac myocytes with sympathetic ganglion. After 3 days of growth, neonatal rat ventricular myocytes developed a stellate configuration. When sympathetic ganglion were co-cultured with the myocytes for initial 72 hours in culture, axons were readily apparent. Individual axons made direct contact with about 10% of myocytes in each culture. These cultured cells were exposed with or without Daunorubicin 1 μ mol. Apoptosis were detected from 8 hours and peaked at 12 hours after Daunorubicin exposure by TUNEL method and DNA laddering. There were significant differences in the rare of TUNEL positive cells between cultured myocytes and sympathetic innervated cultured myocytes after Daunorubicin. But, only a little percent of apoptosis in cultured myocytes and sympathetic innervated cultured myocytes without Daunorubicin exposure were calculated.Next we evaluated the expression of Bax and p53 which were proto-oncogene and its transcriptional factor in cultured and sympathetic innervated myocytes with or without Daunorubicin. Both cultured and co-cultured cells equally up-regulated at 90 after Daunorubicin exposure. Moreover, we detected the expression of Caspase 3 which was enzyme for DNA fragmentation directly was significantly increased in cultured myocyte at 120 after Daunorubicin exposure as compared with those in sympathetic innervated cultured myocytes. Thus, sympathetic innervation could be protected apoptosis in cardiac myocyte by Anthracyclines.