Determining the effects of ischaemia and reperfusion in the intact heart using multi-photon microscopy

We are using a powerful imaging technique called multi-photon microscopy to see what happens inside a rat heart during a simulated heart attack.

Heart disease is a leading cause of death in the UK. After a heart attack, surgeons work to restore blood flow to the heart, but paradoxically this can actually cause further damage to the heart. This may be due to an excess of calcium, or to damaging by-products of oxygen metabolism, but it has been difficult to measure these factors in the intact heart. We are using multi-photon microscopy to generate high resolution images of the cells inside rat hearts. We maintain these hearts alive for several hours after removal from a terminally anaesthetized rat, and simulate a heart attack by temporarily cutting off their oxygen supply.

By directly measuring the changes occurring in the heart over time, we hope to be able to determine what makes them susceptible to injury. Armed with this important information, future research will be directed at protecting these cells, and thus, the heart, from lethal injury during heart attacks.

Myocardial infarction causes the death of cardiomyocytes by causing the accumulation of calcium and oxidative stress in particular, which lead to opening of the mitochondrial permeability transition pore early during reperfusion. Although it is increasingly recognized that the microvasculature is damaged during ischaemia and reperfusion, its contribution to the formation and expansion of the infarct is not known. This is largely because of the difficulty of noninvasively studying the events occurring in the heart at the cellular or subcellular level. The newly developed technology of multiphoton microscopy is capable of visualizing living cells within an isolated perfused heart with much greater resolution than was previously capable with standard confocal microscopy. For example, using this technique it is possible to observe mitochondria during hypoxia and reoxygenation as they depolarize due to opening of the mitochondrial permeability transition pore. This provides the exciting potential to visualize changes in calcium, reactive oxygen species, and mitochondrial function in the intact heart, and to determine the contribution of endothelial damage to these factors. We have obtained preliminary data verifying that it is possible to visualize changes in the mitochondria of live cells within the isolated perfused rat heart. By perfusing with different fluorescent indicators it is possible to visualize changes in calcium levels (fluo4-AM or rhod2-AM), reactive oxygen species production (DCF-DA), and mitochondrial function (TMRM or autofluorescence from NAD(P)H). Additionally, we have obtained high resolution images of the microvasculature, and using techniques described within, will examine the effect of simulated ischaemia and reperfusion on vascular function (i.e.: permeability, tone, and nitric oxide production), and determine if these precede the death of the cardiomyocytes they supply. On the basis of the information obtained from these experiments, future work will be directed specifically at developing methods to protect the cell type most sensitive to the effects of ischaemia and reperfusion.