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Updated: Jul 25, 2026
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion
Derek J Hausenloy1, Michael R Duchen, Derek M Yellon
1The Hatter Institute and Centre for Cardiology, University College London Hospitals and Medical School, Grafton Way, WC1E 6DB London, UK.
Objective:
The opening of the mitochondrial permeability transition pore (mPTP) in the first few minutes of post-ischaemic reperfusion is a critical determinant of reperfusion-induced cell death. We hypothesised that the novel immunosuppressant, sanglifehrin-A (SFA), given at the time of reperfusion, protects the myocardium from ischaemia-reperfusion injury, by suppressing mPTP opening.
Methods:
Isolated perfused rat hearts were subjected to 35 min ischaemia/120 min reperfusion, and were treated with (1) SFA (1.0 microM) or (2) DMSO vehicle for the first 15 min of reperfusion or (3) SFA (1.0 microM) after the first 15 min of reperfusion. We examined the effect of SFA on mPTP opening directly, using a myocyte model of oxidative stress. Laser illumination of adult rat myocytes loaded with the fluorophore, TMRM, generates oxidative stress, which induces mPTP opening (represented by mitochondrial membrane depolarisation) followed by rigour contracture.
Results:
In the isolated perfused heart model, SFA, given during the first 15 min of post-ischaemic reperfusion, reduced the infarct-risk volume ratio from 43.9+/-2.5% in the control group to 23.8+/-4.2% with SFA (p=0.001). However, when SFA was given after the first 15 min of reperfusion, there was no change in infarct size (43.8+/-5.7% with SFA vs. 43.9+/-2.5% in control; p=NS), suggesting that SFA has to be present during the first 15 min of reperfusion to induce protection. In the isolated adult myocyte model, SFA was shown to inhibit mPTP opening in the setting of oxidative stress, represented by an increase in the ROS threshold required to induce: mitochondrial membrane depolarisation (from 269+/-21 to 777+/-100 s; p<0.001) and rigour contracture (from 613+/-14 to 1329+/-129 s; p<0.001).
Conclusions:
Inhibiting mPTP opening during the first few minutes of reperfusion, using sanglifehrin-A, limits infarct size and protects myocytes from oxidative stress.
Insights
Sanglifehrin-A (SFA) limits infarct size by suppressing mitochondrial permeability transition pore (mPTP) opening during early reperfusion. This novel immunosuppressant protects the myocardium from ischaemia-reperfusion injury when administered within the first 15 minutes of reperfusion.
Area of Science:
- Cardiology
- Cell Biology
- Pharmacology
Background:
- Reperfusion-induced cell death is a major concern following ischaemia.
- Mitochondrial permeability transition pore (mPTP) opening during early reperfusion is a key factor in cell death.
- Novel therapeutic strategies are needed to mitigate ischaemia-reperfusion injury.
Purpose of the Study:
- To investigate the protective effects of sanglifehrin-A (SFA) against myocardial ischaemia-reperfusion injury.
- To determine if SFA suppresses mPTP opening during reperfusion.
- To assess the optimal timing for SFA administration to achieve cardioprotection.
Main Methods:
- Isolated perfused rat hearts underwent 35 min ischaemia followed by 120 min reperfusion.
- SFA was administered during the first 15 min of reperfusion or after 15 min.
- The effect of SFA on mPTP opening was directly assessed in a myocyte model of oxidative stress.
Main Results:
- SFA administration during the first 15 min of reperfusion significantly reduced infarct size (23.8% vs. 43.9% in controls).
- Delayed SFA administration (after 15 min) did not alter infarct size.
- SFA inhibited mPTP opening in myocytes, increasing the oxidative stress threshold for mitochondrial depolarisation and rigour contracture.
Conclusions:
- Administering SFA within the first 15 minutes of reperfusion limits infarct size.
- SFA protects the myocardium from ischaemia-reperfusion injury by inhibiting mPTP opening.
- SFA demonstrates potential as a therapeutic agent for managing myocardial ischaemia-reperfusion injury.
