METABOLIC HETEROGENEITY IN ISCHEMIA REPERFUSION INJURY: THE INSIDE STORY

It is well established that ischemia reperfusion (IR) injury can lead to life-threatening arrhythmias. Our group introduced the theory of metabolic sinks as a novel cause of arrhythmogenesis in a heart exposed to IR injury. Metabolic sinks are clusters of myocytes in which ischemia-induced reduction of ATP:ADP ratio increases open probability of sarcolemmal ATP-sensitive K+ channels. This in turn leads to hyperpolarization of membrane potential that reduces or ablates electrical excitability within the affected region of myocardium. To date, studies of metabolic sinks in intact hearts have been largely limited to use of fluorescent indicators to image mitochondrial membrane potential on the epicardial surface. This has revealed the existence of spatio-temporally evolving regions of myocardium within which mitochondrial membrane potential is depolarized, and whose presence influences electrical conduction and action potential duration. In order to explore the three-dimensional structure of metabolic sinks within the myocardium, we have developed a protocol for labeling an intact guinea pig heart exposed to IR injury and imaging any portion of the labeled heart using a custom designed automated volume imaging microtome (AVIM) to overcome the limited imaging field of laser scanning microscopy (LSM). Our AVIM is composed of low-cost components that can be easily installed and removed from a shared microscope. We have developed an open-source software signal processing pipeline to correct for imaging artifacts inherent to LSM and effectively reconstruct the acquired image volumes. Using this approach, we show that hearts undergoing reperfusion arrhythmias have an endocardium containing mostly depolarized mitochondria, with an abrupt transition to repolarized mitochondria in mid-myocardial to epicardial regions. Hearts not exhibiting reperfusion arrhythmias show a much more uniform distribution of depolarized mitochondria as a function of transmural location. These results show different stereotypical patterns of mitochondrial depolarization that are correlated with the presence and absence of reperfusion arrhythmias