TNF-α induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.

ABSTRACT

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.