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Updated: Oct 28, 2025
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic
Yi Song1, Hang Xing1, Yixin He1
1Division of Cardiothoracic Surgery, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
Objective:
Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury.
Methods:
Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 μM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively.
Results:
Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 μM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 μM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05).
Conclusions:
Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.
Insights
Mito-Tempo protects against heart injury by reducing oxidative stress and improving blood vessel function in both diabetic and non-diabetic mice. This antioxidant enhances endothelial function and potassium channel activity after ischemia-reperfusion.
Area of Science:
- Cardiovascular Physiology
- Mitochondrial Biology
- Endothelial Function
Background:
- Cardioplegic ischemia-reperfusion and diabetes mellitus are linked to coronary endothelial dysfunction.
- Oxidative stress, particularly from mitochondrial reactive oxidative species, contributes to this injury.
- Small conductance calcium-activated potassium channels (SK channels) are implicated in endothelial dysfunction.
Purpose of the Study:
- To investigate if the mitochondria-targeted antioxidant Mito-Tempo can protect coronary small conductance calcium-activated potassium channels and endothelial function.
- To determine the protective effects of Mito-Tempo against cardioplegic ischemia-reperfusion-induced injury in a mouse model with and without diabetes mellitus.
Main Methods:
- Isolated small coronary arteries and endothelial cells from diabetic and non-diabetic mice.
- Utilized a cardioplegic hypoxia and reoxygenation model, treating cells with or without Mito-Tempo.
- Assessed microvascular function via myography, K+ currents using patch clamp, and measured Ca2+, mitochondrial reactive oxidative species, and SK protein expression.
Main Results:
- Cardioplegic hypoxia and reoxygenation impaired SK channel activity, increased Ca2+ and mitochondrial reactive oxidative species in both groups.
- Mito-Tempo treatment significantly reduced Ca2+ and mitochondrial reactive oxidative species overload.
- Mito-Tempo enhanced coronary relaxation responses and SK channel currents in both diabetic and non-diabetic mice.
Conclusions:
- Mito-Tempo administration improves endothelial function and SK channel activity following cardioplegic ischemia-reperfusion.
- These improvements may underlie Mito-Tempo's enhancement of endothelium-dependent vasorelaxation.
- Targeting mitochondrial reactive oxidative species with Mito-Tempo offers a potential therapeutic strategy for protecting the coronary endothelium.
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