Cluster Project 2

Metabolic Mechanisms of Sudden Cardiac Death in the Failing Heart

Aim 2.1: Measure changes in the expression of transcripts and proteins involved in mitochondrial function in both normal and failing canine heart tissue. This aim will quantify the altered metabolic state of failing canine ventricular myocytes by measuring and comparing levels of transcripts and proteins relevant to mitochondrial function, including electron transport, ROS production and scavenging. The proteomic data will be used to adjust the levels of mitochondrial proteins with function described in the normal and failing canine myocyte models to be developed in CP3 Aim 3.3. The functional consequences of these changes will be explored using a combination of metabolic control analysis and direct model simulation and model predictions regarding susceptibility to metabolic oscillations will be made. These predictions will be tested experimentally in Aim 2.2.

Aim 2.2: Test the hypothesis that ventricular myocytes isolated from failing hearts exhibit increased predisposition to oscillations of ΔΨm, ROS release, oscillatory activation of IKATP and membrane potential. The models developed in CP3 Aim 3.3, using the data of Aim 2.1 above, will be used to predict whether or not ventricular myocytes are more susceptible to metabolic oscillation in the setting of heart failure than under normal conditions and to determine the mechanisms underlying any differences in ROS-induced oscillations. Aim 2.2 will then test these model predictions by measuring the electrophysiological responses of cells isolated from normal and failing canine hearts to interventions designed to induce mitochondrial oscillations and ROS release. Isolated ventricular myocytes will be treated with diamide or a laser flash to induce mitochondrial depolarization, ROS release and oscillatory changes in ΔΨm and cellular ATP. Susceptibility to oscillations will be contrasted in normal and failing cells by comparing the latency to oscillations in response to localized ROS release induced by a laser flash, and the concentration of diamide (EC50) required to induce oscillations. Whole-cell patch clamp recording will be performed to monitor changes in the cellular AP and CaTs induced by diamide and flash. The nature of electrophysiological and metabolic coupling observed in normal and failing canine myocytes will be understood through iterative collection of experimental data, testing the data against model predictions obtained in CP3, and using the model results to refine experimental approaches.

Aim 2.3: Test the hypothesis that in the remodeled failing heart, an exaggerated response to metabolic stress induces mitochondrial membrane potential oscillations that lead to formation of metabolic sinks and ventricular arrhythmias. High-resolution three-dimensional anatomical reconstructions of control and failing canine LV tissue wedges will be generated (Project 1, Aim 1.3) and used in constraining preparationspecific, anatomically and biophysically detailed models of the same canine wedges that are studied experimentally. These models will be used to predict whether or not failing canine myocardium is more or less susceptible to the formation of metabolic sinks, to determine the critical metabolic sink tissue mass required for induction of reentry and to investigate tissue properties that influence these behaviors, and to predict whether arrhythmia in metabolically-stressed hearts results from reentry about the in-excitable core of the metabolic sink and/or regional dispersion of APD and repolarization. The model predictions will tbe tested experimentally using complementary methods of inducing metabolic stress, including ischemia and reperfusion, exposure to mitochondrial uncouplers and thiol cross-linking reagents to perturb mitochondrial function in normal and failing canine LV tissue wedges. Optical APs, CaTs, ΔΨm and frequency of spontaneous arrhythmias will be compared in control and failing wedges subjected to metabolic stress. We will initially utilize ischemia/reperfusion as a reproducible, reversible and clinically relevant metabolic stress. The key molecular mediators of the electrophysiological responses will be studied by inhibition or activation of IMAC (4’-Cl diazepam or FGIN-1-27 respectively), inhibition of the permeability transition pore (PTP; cyclosporin A) or inhibition of the sarcolemmal IKATP (glibenclamide). Experimental measurements will be compared directly with those predicted using the preparation-specific models of CP3 Aim 3.4, and the results will be used to iteratively refine the models and to suggest new hypotheses.