Cardiac Troponin I A164H and pH-Dependent Inotropy

Abstract

Ischemic heart disease is the most common cause of mortality in the world. To develop novel therapeutic strategies and gain mechanistic insight we are using time-resolved fluorescence measurements to detect structural changes within the cardiac troponin complex that occur during acute ischemic insult. During myocardial ischemia, penultimate to cell death, there is a shift from oxidative to glycolytic metabolism resulting in an acidification that severely impairs sarcomere function. In cardiac myocytes, this decrease in pH uncouples calcium homeostasis from force-generation. We and others have shown that the neonatal cardiac isoform of TnI, ssTnI, confers pH-insensitivity in this regard compared to the adult cTnI isoform. However ssTnI confers deleterious effects of impaired relaxation in the adult myocyte. Alignment and functional studies have demonstrated that this pH-insensitivity is derived from ssTnI residue H132. Introduction of a histidine at the cognate position in cTnI (A164H) mitigates the pH-sensitivity of the calcium-force relationship in cardiac myocytes while retaining relaxation enhancement via the N-term domain relative to ssTnI. We are establishing a time-resolved fluorescence methodology for detecting alterations in the calcium sensitivity of the thin filament during ischemia. We have engineered a single cysteine mutation for labeling with environmentally sensitive fluorophores designed to detect Ca2+ and pH-sensitive structural changes in cTnI and cTnC. We will discuss progress using this approach to interrogate troponin function in ischemia mimetic conditions.