New ways of imaging the cardiac myocyte436Slow calcium waves and redox changes precede mPTP opening occur during hypoxia and reoxygenation in the intact heart

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Abstract

Aim

Opening of the mitochondrial permeability transition pore (mPTP) occurs during cardiac ischemia and reperfusion and is an irreversible step on the pathway towards cardiomyocyte death. In isolated mitochondria, mPTP opening is triggered by calcium overload facilitated by oxidative stress, although the chronology has been suggested to be different in isolated cells. Our objective was to establish the events that cause mPTP opening in the intact heart.

Methods

We performed multiphoton imaging of Langendorff-perfused mouse hearts expressing an inducible, Ca2+-sensitive reporter (GCaMP2), to examine the response to a hypoxia-reoxygenation protocol of individual cardiomyocytes within an intact, perfused heart.

Results

We found that during reperfusion, spontaneous calcium waves arose and propagated across multiple cells at 3.3 μm/s. In cells in which [Ca2+] returned to normal there was no effect on mitochondrial membrane potential, measured using the dye TMRM. However, in ~ 56% of cells the calcium waves were followed after 100 ± 27 s (N=10 hearts) by loss of TMRM fluorescence due to opening of the mPTP. During reoxygenation, co-ordinated redox changes also occurred across large regions, and preceded mPTP opening on average by 122 ± 38 s (N=9 hearts). Significantly fewer calcium waves resulted in mPTP opening in the presence of mPTP inhibitor cyclosporin A, or mitochondrial-targeted ROS scavenger, MitoQ. Only MitoQ, however, reduced the number of Ca2+ waves.

Conclusion

These experiments define the spatiotemporal relationship between changes in Ca2+, redox state, and mPTP opening during reoxygenation in the intact heart. Tissue oxidation coincident with localized calcium waves together conspire to cause mPTP opening and subsequent cell death.

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