Detection of early ischemic damage by analysis of mitochondrial function in skinned fibers

The skinned fibers technique was applied for studies of the effects of global acute ischemia (1 h at 37 degrees C) and long time (15 h) hypothermic (4 degrees C) preservation of isolated rat hearts under different conditions (immersion or low-flow perfusion) on mitochondrial function in the cells in vivo. Skinned fibers were obtained by using saponin for permeabilization of the sarcolemma in separated fiber bundles cut from left ventricle. The experimental protocol of the respiration rate determination included a cytochrome c test to check the intactness of the outer mitochondrial membrane. The apparent Km for ADP and the effect of creatine on the mitochondrial activity were also evaluated in these permeabilized fibers, taken from different groups of hearts. The preservation of low-flow perfused hearts resulted only in a slight decrease of creatine (20 mM) stimulated respiration at 0.1 mM ADP. The fibers from ischemic hearts or from hearts preserved by immersion showed a decrease of the apparent K-m for ADP, and a complete loss of the stimulatory effect of creatine. Tn these fibers, we could observe that the outer mitochondrial membrane was damaged. In conclusion, the results of this study show that assessment of mitochondrial parameters sensitive to organelles swelling - intactness of outer membrane and functionally coupled creatine kinase reaction - are the most sensitive indicators of early hypoxic or ischemic damage to mitochondria. Their determination in biopsy samples could be used for evaluation of the efficiency of the cardiac protection in heart surgery.