Extracellular mitochondrial release from failing hearts impairs skeletal muscle

Background In the progression of heart failure, concomitant skeletal muscle disorders contribute to the decline in patients’ activity of daily living. To preserve volume and quality of skeletal muscle in patients with heart failure, only cardiac rehabilitation is currently carried out. The mechanisms of skeletal muscle damage in heart failure have not been elucidated. Mitochondria are key organelles of energy metabolism in cardiac myocytes as well as critical factors of programmed cell death in heart failure. Recently, extracellular mitochondrial release and their vascular endothelial impairment were reported after brain attack. Purpose We investigated whether mitochondria are released from cardiac myocytes in heart failure, and if so, the roles of extra-cellular mitochondria on skeletal muscle. Methods Rat cardiac ventricular H9C2 cells and differentiated skeletal muscle C2C12 cells were cultured. H9C2 were exposed to angiotensin II (100 nM) and norepinephrine (1 μM) for 24 hours. After 24 hours, culture media were corrected, and floating debris and cells were removed by centrifugation. Then, supernatant was centrifuged again at 17,000 g and pellet was resuspended using phosphate buffered saline, as small molecule-enriched fraction (SMF). Content of mitochondrial Tom20 protein in SMF was assessed by Western blot analysis. Then, C2C12 skeletal muscles were incubated with medium containing SMF for 48 hours. Injury of skeletal muscle were assessed by LDH activity in culture medium, MMT assay, and Myo D mRNA expression. To examine the effect of extracellular mitochondria on skeletal muscle injury, C2C12 was incubated with mitochondria-reduced SMF by immunoprecipitation. In addition, in vivo experiments male Wistar rats were subjected to coronary ligation to introduce myocardial infarction (MI). 2 to 3 months later, plasma was corrected from MI and sham-operated rats and SMF were obtained. Then, SMF was administrated to C2C12 cells. Results Nether angiotensin II nor norepinephrine increase LDH activity in H9C2 culture medium after 24 hours. Angiotensin II, but not norepinephrine, increased mitochondrial Tom20 content by 50% in SMF. AII-treated H9C2-derived SMF (AII SMF) significantly increased LDH activity in C2C12 culture media by 15% and decreased MMT absorbance by 8%, indicating AII SMF induced skeletal muscle injury. In addition, AII SMF significantly suppressed MyoD mRNA expression in C2C12 cells by 23%. Reduction of mitochondria in AII SMF ameliorated C2C12 injury, examined by LDH activity and MTT assay. SMF obtained from plasma of MI rats contained more Tom20 protein and also increased LDH activity in C2C12 culture medium compared to SMF obtained from plasma of sham-operated rats. Conclusion These results indicate that cardiac myocytes release mitochondria without disruption of plasma membrane in heart failure and these myocyte-derived mitochondria impair skeletal muscle cells.