RESUMO
Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. SpragueDawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin6 (IL6) and tumor necrosis factorα (TNFα). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylatedDrp1 (pDrp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNFα (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and pDrp1 Ser616. Following TNFα treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNFαinduced increase in pDrp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y27632 and fasudil may also attenuate the TNFαinduced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNFα is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.
Assuntos
Dinaminas/imunologia , Inflamação/patologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Linhagem Celular , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Masculino , Mitocôndrias Cardíacas/imunologia , Miocárdio/imunologia , Miocárdio/patologia , Miócitos Cardíacos/imunologia , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
In the present study, the alterations in uncoupling protein 2 (UCP2) expression following hypothermic preservation in rat hearts were investigated. Isolated rat hearts were preserved in Celsior solution for 312 h followed by 60 min of reperfusion. The cardiac function was evaluated using the Langendorff perfusion system. UCP2 and silent mating type information regulation 2 homolog 1 (SIRT1) proteins were detected by western blot analysis. The ATP production and mitochondrial reactive oxygen species (ROS) levels were assessed. Subsequent to preservation in icecold Celsior solution for 312 h, the UCP2 protein expression in rat hearts was observed to increase in a timedependent manner. The UCP2 inhibitor genipin inhibited the hypothermic preservationinduced cardiac dysfunction, prevented a decline in ATP production induced by 9 h of preservation, however had no effect on the hypothermic preservationinduced increase in mitochondrial ROS levels. Compared with the control group, the SIRT1 protein expression in rat hearts reduced following hypothermic preservation. Compared with the 9h preservation group, Celsior solution supplemented with the SIRT1 activator resveratrol (20 or 40 µmol/l) inhibited UCP2 protein overexpression, prevented the decline in ATP production and resulted in an improvement cardiac function. The SIRT1 inhibitor EX527 abolished the resveratrolinduced inhibition of UCP2 overexpression and cardiac protection in the hypothermic preserved rat heart. These observations suggest that downregulation of UCP2 expression in the hypothermic preserved rat heart in part initiated the protective mechanism via the SIRT1 pathway.