Length-Dependent Activation in Human Myocardium

This collaborative project integrates the skills and resources of five investigators to advance understanding of a cellular-level mechanism that underpins the Frank-Starling relationship. Specifically, the project focuses on length-dependent activation, defined as the increased maximum force and Ca2+ sensitivity of contraction induced by myocardial stretch. The research builds on recent discoveries relating to the myosin super-relaxed (also known as Interacting Heads Motif) state and targets dynamic OFF/ON transitions in thick filament structure. The Frank-Starling mechanism is impaired in patients who have heart failure. Some of the 6 million Americans afflicted with this condition are carrying a mutation associated with a cardiomyopathy, but penetrance varies dramatically and genetics rarely influences treatment. For example, the University of Kentucky is currently performing 1% of worldwide cardiac transplants but its clinicians “treat phenotype, not genotype”. Most candidates for transplant are described simply as having ischemic heart failure (that is, heart failure subsequent to an infarction) or non-ischemic heart failure (everything else). We have analyzed myocardial samples procured from organ donors and transplant recipients. Our preliminary data suggest that length-dependent changes in Ca2+ sensitivity are eliminated in myocardium from patients who have non-ischemic heart failure but preserved in organ donors and patients who have ischemic heart failure. New computer modeling predicts that these functional changes may reflect destabilization of the myosin OFF state in patients who have non-ischemic heart failure. This hypothesis is supported by pilot experiments that use fluorescent polarization techniques to assess OFF/ON transitions in the human samples. Based on these data, we designed peptides to stabilize or destabilize the myosin OFF state. Initial results suggest that the stabilizing peptides reduce the Ca2+ sensitivity of contraction while de-stabilizing peptides increase Ca2+ sensitivity in a length-dependent manner that matches the predictions of the computer model. Our research plan builds on these exciting preliminary data and integrates multiple biophysical techniques to test the global hypothesis that length-dependent activation is reduced in patients who have non-ischemic disease because cardiac thick filaments are biased towards the myosin ON state. Aim 1: Test the hypothesis that length-dependent changes in Ca2+ sensitivity are reduced in myocardium from patients who have non-ischemic heart failure. Quantify length-dependent activation in multicellular preparations (Tanner) and single myocytes (McDonald) isolated from organ donors and patients who have ischemic or non-ischemic heart failure. Aim 2: Test the hypothesis that the OFF state of the thick filament is destabilized in myocardium from patients who have non-ischemic heart failure. Fluorescent polarization measurements in multicellular preparations (Sun) and pulse-chase mantATP experiments in single myocytes (McDonald) from organ donors and patients. Aim 3: Target OFF/ON transitions to manipulate the Ca2+ sensitivity of human myocardium. Contractile assays (Tanner, McDonald) testing peptides designed to stabilize or destabilize the myosin OFF state (Root). Additional studies explore whether targeting OFF/ON transitions can improve lengthdependent activation in vitro in myocardium from patients with non-ischemic heart failure. Aim 4: Use computer modeling to predict how perturbing OFF/ON transitions impacts hemodynamics. Multiscale simulations (Campbell) supported by experiments using living human cells (Campbell/Tanner).