Abstract P088: The Rate of Inorganic Phosphate Release in Contracting Rat Trabeculae Is Independent of Calcium After Stretch

Abstract

We aim to reveal the molecular mechanism underlying the force response to stretch in the heart by examining the dependence of the rate of inorganic phosphate (P i ) release in response to stretch on the degree of calcium activation. Permeabilized trabeculae of rat heart are activated by photolytic release of ATP, at 20°C, in the presence of saturating (32µM), or half-saturating (1µM), calcium concentrations, at an initial sarcomere length of 1.9 or 2.1 µm. For trabeculae with an initial sarcomere length of 1.9 µm, the trabeculae are stretched to 2.1 µm during the isometric plateau, held at a constant length and returned to their original length. The rate of P i release is determined by the fluorescence change associated with phosphate binding to a fluorescently-labelled phosphate binding protein diffused into the preparation. At an initial sarcomere length of 1.9µm, the isometric force at full activation was 51 kN.m −2 compared to 26kN.m −2 at half-maximal activation. During the isometric phase, P i release is at a steady rate of 8.2 and 4.8 s −1 (assuming a myosin head concentration of 120µM) at 32 and 1µM calcium, respectively. During stretch, the rate of P i release decreases markedly at both activation levels, to1.0 and 1.4s −1 respectively. Activation from an initial sarcomere length of 2.1µm produced an isometric force of 65.5 kN.m −2 with a steady P i release rate of 8.1 s −1 at maximal activation, and an isometric force of 49.2 kN.m −2 with a steady P i release rate of 4.8 s −1 at half-maximal activation. The rates of P i release during stretch and after the end of the stretch at low activation are equal to that at high activation indicating stretch-induced activation which occurs for stretches applied prior to activation, and during activation. The results demonstrate that stretch has a dramatic effect causing an immediate reduction in the rate of P i release. Stretch contributes to activation of the trabecula's actomyosin ATPase in a calcium-independent manner, although the effect is more marked at low activation levels. Thus a direct effect of stretch on thin filament activation contributes to the Frank-Starling law.