Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C129
Slow, spontaneous calcium waves occur during reperfusion after cardiac ischaemia, and precede opening of the mitochondrial permeability transition pore.
S. M. Davidson1, M. M. Duchen2, D. M. Yellon1
1. Hatter Institute, Medicine, University College London, London, United Kingdom. 2. Cell and Developmental Biology, University College London, London, United Kingdom.
For many years, progressive calcium overload during ischaemia has been understood to be one of the major contributing factors leading to cell death during reperfusion. However, measuring calcium in the intact heart is technically challenging and has been largely restricted to spatially averaged measurements that obscure the response of individual cells. METHODS: We have performed two-photon imaging of isolated, Langendorff perfused hearts from transgenic mice expressing the recently developed Ca2+-sensitive fluorescent protein, GCaMP2, to examine the response of individual cardiomyocytes within an intact, perfused heart to a hypoxia-reoxygenation protocol. Hearts were removed after terminal anaesthesia using sodium pentobarbital administrated i.p. In order to relate Ca2+ increase to opening of the mitochondrial permeability transition pore (mPTP), hearts were perfused with the fluorescent dye TMRM (100 nM), which loads into mitochondria with an intact mitochondrial potential but is lost upon mPTP opening. RESULTS: During reoxygenation, we observed transient calcium waves that originate in apparently healthy cells and spread relatively slowly (at a rate of 3.3 ± 0.3 μm / s ; n=26 events) up to a distance of several cells through the myocardium. This is much slower than spontaneous Ca2+ waves (typically ~100 μm / s) that have previously been observed with localization restricted to within individual injured myocytes with Ca2+ overload, and suggests a distinct mechanism of generation. In cells in which [Ca,2+]cytosol returns to normal there is no effect on TMRM fluorescence. However, in ~ 56% of cells the calcium waves precede opening of the mPTP by 100 ± 27 s (n=9 events), an irreversible step towards myocyte death. In separate hearts, a loss of NAD(P)H autofluorescence was observed to precede mPTP opening in cardiomyocytes by a similar amount, suggesting the Ca2+ waves and loss of NAD(P)H signal may be causally related. CONCLUSION: These observations suggest that mPTP opening may be caused by spatially restricted, transient increases in [Ca2+]cytosol in the form of waves that are able to spread between cells, possibly contributing to the “wave-front” phenomenon of infarct progression.
Where applicable, experiments conform with Society ethical requirements