Aim: Following previous studies on the ischemia-induced adaptive changes in human cardiac mitochondria, we examined in the present paper the interaction between nitric oxide-induced (NO) partial inhibition of Cyt. c oxidase (Cyt.OX) and mitochondrial encoded subunit 2 expression. Aim of the study was to investigate specific stages of the biochemical and molecular cascade which takes place in cytoprotective mechanisms of ischemic and reperfused cardiac cell.

Methods: We examined human left ventricle samples obtained from 20 patients undergoing elective valve surgery before aortic cross-clamping, 20+/-2 min (prolonged ischemia), 58+/-5 min after cross-clamping (intermittent ischemia) and 21+/-4 min after reconstitution of coronary blood flow (reperfusion). Cyt.OX activity was determined by spectrophotometric method and adenosine triphosphate (ATP) content using bioluminescent assay. Malondialdehyde (MDA) assumed as reactive oxygen species (ROS) generation marker was determined by high-performance liquid chromatography method. On the same cardiac samples mitochondrial encoded Cyt.OX subunit 2 expression was examined by immunoblot analysis and blu native gel electrophoresis method. Statistical study of obtained data was performed using repeated measures analysis of variance (ANOVA).

Results: Prolonged as well intermittent ischemia caused reduction of Cyt.OX activity and ATP, a moderate accumulation of ROS and down-regulation of Cyt.OX subunit 2. When reperfused the cardiomyocytes showed a progressive increase of Cyt.OX activity, ATP pools and Cyt.OX subunit 2 expression. ROS generation was significantly increased by the rapid oxygen re-immission in the cardiac cell.

Conclusions: These data confirm the suggestion that prolonged as well as intermittent ischemia induces activation of cytoprotective mechanisms crucial for cardiac cell survival. Indeed, co-ordinated down-regulation of Cyt.OX activities, ATP pools and mitochondrial encoded Cyt.OX subunit 2 are in favour of an ischemia-activated adaptive mechanism leading to transient and reversible oxidative injury. This observation is confirmed by reduction of apoptosis molecular markers and by complete recovery of mitochondrial oxidative activities in reperfused cardiac tissue.