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Updated: May 20, 2025
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
"Villains" Turning Good: Antimycin A and Rotenone, Mitochondrial Respiratory Chain Inhibitors, Protect H9c2 Cardiac
Kyriaki Zikaki1, Eleni Kiachaki1, Catherine Gaitanaki1
1Section of Animal and Human Physiology, Faculty of Biology, School of Science, National and Kapodistrian University of Athens, University Campus, Ilissia, 15784 Athens, Greece.
Abstract:
Mitochondria are the powerhouses of cells, also involved in ROS (reactive oxygen species) generation and cellular death regulation. Thus, several diseases are associated with mitochondrial impairment, including cardiovascular disorders (CVDs). Since CVDs are currently the leading cause of death worldwide, it is very important to evaluate targeting mitochondrial effectors in clinical treatment protocols. Hence, in the present study, antimycin A and rotenone, established inhibitors of the mitochondrial electron transfer chain, were shown to halt apoptotic death induced by curcumin (50 μM) and sorbitol (0.5 M), in H9c2 cardiac cells. In particular, immunoblotting analysis revealed that they totally abolished PARP [poly(ADP-ribose) polymerase] proteolysis, under these conditions. This finding was accompanied by an enhancement of cell viability, recovery of mitochondria networks' integrity, suppression of cytochrome c release into the cytoplasm, and reversal of chromatin condensation. Chelating extracellular calcium (with EGTA) further enhanced the beneficial impact of antimycin A and rotenone on curcumin- or sorbitol-treated H9c2 cells viability. Of interest, the phosphorylation of eIF2α, indicative of the onset of the pro-survival Integrated Stress Response (IRS), was sustained under these conditions. Overall, our data highlight the anti-apoptotic effect of these compounds, unmasking their potential as mediators in novel therapeutic interventions against mitochondria-associated cardiac dysfunction.
Insights
Mitochondrial electron transfer chain inhibitors, antimycin A and rotenone, prevent cell death in cardiac cells. These compounds offer potential therapeutic benefits for cardiovascular disorders by protecting mitochondria.
Area of Science:
- Cell Biology
- Cardiovascular Science
- Mitochondrial Biology
Background:
- Mitochondria play crucial roles in cellular energy production, reactive oxygen species (ROS) generation, and regulating cell death.
- Mitochondrial dysfunction is implicated in various diseases, notably cardiovascular disorders (CVDs), the leading global cause of mortality.
- Targeting mitochondrial pathways presents a promising strategy for novel therapeutic interventions in CVDs.
Purpose of the Study:
- To investigate the anti-apoptotic effects of antimycin A and rotenone on H9c2 cardiac cells.
- To explore the potential of these mitochondrial electron transfer chain inhibitors in mitigating cardiac dysfunction.
Main Methods:
- H9c2 cardiac cells were treated with curcumin or sorbitol to induce apoptosis.
- Apoptosis was assessed by monitoring PARP proteolysis, cell viability, mitochondrial network integrity, cytochrome c release, and chromatin condensation.
- Inhibitors of the mitochondrial electron transfer chain (antimycin A, rotenone) and extracellular calcium chelator (EGTA) were employed.
Main Results:
- Antimycin A and rotenone effectively halted curcumin- or sorbitol-induced apoptotic death in H9c2 cells.
- These inhibitors prevented PARP proteolysis, enhanced cell viability, preserved mitochondrial networks, suppressed cytochrome c release, and reversed chromatin condensation.
- EGTA treatment potentiated the protective effects of antimycin A and rotenone, while sustaining the Integrated Stress Response (IRS) via eIF2α phosphorylation.
Conclusions:
- Antimycin A and rotenone exhibit significant anti-apoptotic properties in cardiac cells.
- These compounds demonstrate potential as therapeutic agents against mitochondria-associated cardiac dysfunction.
- Modulating mitochondrial function via electron transfer chain inhibition offers a novel approach for treating cardiovascular diseases.
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