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Updated: Aug 8, 2026
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Mitochondrial genome involvement in ischemia/reperfusion-induced adaptive changes in human myocardial cells
G C Corbucci1, B Lettieri, C Luongo
1Departement of Anesthesia and Resuscitation University of Cagliari, Cagliari, Italy. corbucci@unica.it
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.
Insights
Cardiac ischemia triggers adaptive responses involving nitric oxide (NO) and mitochondrial function. These changes, including Cyt. c oxidase (Cyt.OX) regulation, protect heart cells from oxidative injury during and after ischemic events.
Area of Science:
- Cardiovascular Biology
- Mitochondrial Medicine
- Cellular Adaptation
Background:
- Ischemia-induced adaptive changes in human cardiac mitochondria are critical for cell survival.
- Nitric oxide (NO) plays a role in modulating mitochondrial function during cardiac stress.
Purpose of the Study:
- To investigate the interaction between NO-induced partial inhibition of Cytochrome c oxidase (Cyt.OX) and mitochondrial encoded subunit 2 expression.
- To elucidate the biochemical and molecular cascade in cytoprotective mechanisms of ischemic and reperfused cardiac cells.
Main Methods:
- Human left ventricle samples from valve surgery patients were analyzed at different ischemia and reperfusion time points.
- Assessed Cyt.OX activity, ATP content, reactive oxygen species (ROS) generation (malondialdehyde), and Cyt.OX subunit 2 expression.
- Utilized spectrophotometry, bioluminescent assay, HPLC, immunoblot analysis, and blue native gel electrophoresis.
Main Results:
- Prolonged and intermittent ischemia reduced Cyt.OX activity, ATP, and Cyt.OX subunit 2 expression, with moderate ROS accumulation.
- Reperfusion led to increased Cyt.OX activity, ATP, and Cyt.OX subunit 2 expression.
- Rapid oxygen re-introduction during reperfusion significantly elevated ROS generation.
Conclusions:
- Ischemia activates cytoprotective mechanisms involving coordinated down-regulation of Cyt.OX, ATP, and Cyt.OX subunit 2.
- These adaptive changes contribute to transient and reversible oxidative injury, promoting cardiac cell survival.
- Reduced apoptosis markers and full recovery of mitochondrial function post-reperfusion confirm these protective adaptations.
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