Exploring the Link between Ca2+ Signalling and ATP Dynamics in Cardiomyocyte Dysfunction

Abstract

In heart failure the progressive deterioration of cardiac function is linked to Ca2+ handling abnormalities, altered metabolism and increased cardiomyocyte apoptosis. However the causal versus consequential nature of these linked phenomena remains to be determined. Here we aimed to investigate cellular ATP and Ca2+ environments and necrotic and apoptotic cell death in cellular models of Ca2+ dysfunction and metabolic perturbation. We constructed new ATP sensors via fusion of a red fluorescent protein (mCherry) with the pH-independent click-beetle luciferase in both mCherry-Luc and Luc-mCherry configurations. Chimeric proteins exhibited high specific activities of the luciferase partner in vitro (approximately 5 cps/amol protein) and we determined linear fluorescence-luminescence relationships (r2 > 0.88) following their expression in COS-7 cells. The dynamic range of the Luc-mCherry configuration was determined via exposure of COS-7 cells to manoeuvres that depleted ATP (e.g. 2-DOG, FCCP) and under conditions of elevated glucose availability (> 6g/L extracellular). In HL-1 cardiomyocyte monolayers, we monitored cellular ATP environments following pharmacologic manipulation of the amplitude and temporal patterning of Ca2+ signaling (e.g. using ouabain, staurosporine, BAPTA). Ca2+ and ATP environments were monitored through the transitions from spontaneously oscillatory syncytia to non-oscillatory phenotypes. Under these conditions, we measured cellular ATP effluxes and susceptibility to apoptosis using in situ DNA fragmentation and caspase activation assays. This approach reconciles specific perturbations in Ca2+ handling, the cellular ATP environment and phenotypic outcomes and builds a more complete picture of the events that contribute to the early stages of cellular dysfunction in chronic heart disease.