Cluster Project 1

Metabolic Sinks as a Mechanism of Sudden Cardiac Death in the Ischemic Heart

Aim 1.1: Effects of mitochondrial uncoupling on electrical propagation in the GP heart. This aim will test the metabolic sink hypothesis by determining if proton leak (e.g., mitochondrial uncoupling) initiated by a chemical uncoupler (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, FCCP) and the resulting activation of IK_ATP leads to abnormal electrical propagation in intact-perfused guinea pig hearts. The uncoupler will be applied either: a) globally, for a short duration, followed by washout; or b) locally, via regional perfusion or direct injection. Optical mapping of action potentials (APs), Ca2+ transients (CaT) and ΔΨm on the epicardial surface of the guinea pig heart will be performed at high spatial resolution using two-photon excitation of fluorescence and at lower resolution, but with a much more broad field of view using photodiode arrays. These data will be used to test the hypotheses that arrhythmia in metabolically-stressed hearts results from: a) reentry about the in-excitable core of the metabolic sink; and/or b) regional dispersion of APD and repolarization.

Aim 1.2: Tissue localization of mitochondrial depolarization. In order to understand the ways in which metabolic sinks affect electrical propagation in the myocardium, we will determine the region over which metabolic uncoupling occurs by rapidly loading hearts with MitoTracker Red (Invitrogen Corp.) and then fixing them for 2-photon imaging using an automated microtome method (Aim 1.3). Mitotracker Red loads into polarized mitochondria and is retained after fixation and permeabilization (e.g. for co-staining with antibodies). MitoTracker Red emission can be imaged simultaneously with blue (<500nm) or green (500-550nm) emissions for several permutations of structure-function correlation experiments. We will co-stain with Hoescht 33342, which will label cell nuclei with a blue fluorescence emission, to count the number of cells present in each image field regardless of the bioenergetic stain. In this way, regions of metabolic comprise can by reconstructed in three dimensions, and correlated with functional data acquired prior to fixation.

Aim 1.3: Measurement of cardiac tissue structure. The fiber structure of the cardiac ventricles is a major determinant of electrical conduction patterns. Fiber orientation at corresponding points in different hearts is highly variable (Fig. 5, Helm et al – standard deviation can be as much as 20°). Thus, in order to compare model predictions from CP3 directly against experimental data, it is necessary to measure both conduction patterns (Aim 1.1), the location of metabolic sinks (Aim 1.2) and the geometry and fiber structure of each heart. This aim will make high spatial-resolution measurements of GP ventricular structure using a novel two-photon second-harmonic generation (SHG) imaging technique. Canine LV wedges studied in Aim 2.3 will also be reconstructed using SHG imaging. This will be accomplished by fixing tissues after optical mapping and mitochondrial labeling, followed by structural analysis using a combined SHG imaging/integrated microtome technique in this aim. In CP3, these anatomic data will be used to develop GP heart and canine tissue-wedge models that will be used to formulate hypotheses as to how metabolic sinks, layered on the underlying ventricular tissue architecture, contribute to the generation of arrhythmias. These hypotheses will then be tested experimentally.