Heart disease is the leading cause of death in the United States and has been rising dramatically around the world. This is an exploratory application to develop strategies for improving cardiac performance by targeting the contractile apparatus of cardiomyocyte sarcomeres. While the current application focuses on in vitro studies, the long term goal is to enhance systolic function without compromising diastolic function of the heart, and still allow responsiveness to adrenergic stimulation. We will target the cardiac thin filament activation and the actin-myosin `crossbridge' cycle directly, such that cardiomyocyte contraction (but not relaxation) is enhanced without the need for increased intracellular [Ca2+]. To increase thin filament activation at a given [Ca2+] we will replace native troponin C in myofilaments with a mutant TnC (L48Q) that has enhanced Ca2+ binding properties (aim 1). To enhance crossbridge cycling we will increase the cellular production of 2 deoxy-ATP (dATP) via increased expression of the enzyme that converts ATP to dATP in cardiomyocytes (ribonucleotide reductase; RR) (aim 2). We have provided significant data demonstrating these approaches improve contractility in demembranated (skinned) cardiac tissue, without affecting relaxation kinetics or resting stiffness. For the proposed studies we will use a viral transfection strategy to determine if similar increases in cardiac contractility occur in intact adult cardiomyocytes in culture. We will also determine whether these approaches affect sarcomere contractile protein isoform and phosphorylation profiles or the level of intracellular [Ca2+] during stimulated activation and in relaxation. In the second year of the proposal we will begin development of animal models for an expanded proposal to study how these manipulations influence whole heart function in situ and in vitro in normal hearts and under pathological conditions.
Heart failure at its base is a reduction in cardiac myofilament contractility. Most current therapies focus on mechanisms that enhance intracellular Ca2+ during systole which, among other things, can affect diastolic function. To avoid this, our proposal targets myofilaments directly to enhance contraction without the need for increased intracellular Ca2+. ? ? ?
