Cardiovascular disease is the leading cause of death worldwide. An important component of cardiac disease is myocardial ischemia, the pathophysiological condition resulting from the interruption of oxygen and blood supply to cardiac muscle. The necessary remedy for ischemia is re-introduction of blood flow into the ischemic muscle (reperfusion). However, reperfusion causes a divergent effect, salvaging some cells and killing others, known as ischemia/reperfusion (I/R) injury (IRI). In spite of decades of research, the exact mechanisms and pathways by which individual cardiomyocytes die in the aftermath of a heart attack remain poorly understood. Competing theories postulated that either mitochondrial membrane potential collapse due to the mitochondrial permeability transition (MPT), or cell membrane (sarcolemmal) permeabilization (SP) is the pivotal point in the path to cell death. Pathological increases in the cytoplasmic or mitochondrial calcium concentration ([Ca2+]cyto or [Ca2+]mito) have been implicated in SP and MPT, respectively. However, prior studies have not systematically examined the timing and causative relationship between MPT, SP, and intracellular [Ca2+] shifts during real I/R in the proper context of the myocardial syncytium. Driven by specific favored hypotheses, prior studies failed to establish a systemic view of the "death pathway(s)", i.e., a consistent sequence of critical events leading to cellular catastrophe. Lack of such systemic understanding may in part explain the poor outcomes in the majority of clinical trials aimed to reduce the extent of IRI.
This proposal aims to fill these gaps in knowledge by simultaneously tracking critical mitochondrial, sarcolemmal, and Ca2+ events in whole hearts and cell monolayers subjected to I/R. Using multi-channel, live confocal microscopy we will be able to establish the typical sequence(s) of critical events in individual myocytes within a myocardial syncytium. Analyzing spatiotemporal information from at least three different fluorescent indicators (including a genetically encoded indicator of [Ca2+]mito), we will be able to time the events of MPT and SP, as well as abnormal Ca2+ elevations in the cytoplasm and mitochondrial matrix, in the same myocyte. The approach does not favor any specific hypothesis and aims to provide an unbiased, quantitative, and the most systematic to-date description of the cardiomyocyte transition from life to death. Identifying the "point-of-no-return" event in the cell injury during I/R will provide essential information in the quest to identify better therapeutic targets for the improvement of tissue viability after I/R, thus reducing damage from IRI and increasing the survival rate in sudden cardiac arrest.