Modeling Regulation of Mitochondrial Free Ca2+ by ATP/ADP-Dependent Ca2+ Buffering

Abstract

Introduction: Mitochondrial free [Ca2+] ([Ca2+]m) is regulated by cation fluxes through the Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as via Ca2+ buffering by the mitochondrial proteins. However, the regulation of [Ca2+]m via ATP/ADP-dependent dynamic Ca2+ buffering mechanism inside the mitochondrial matrix during transient state-3 respiration is not well known. Methods: To gain a quantitative understanding of this Ca2+ buffering phenomenon, we developed a computational model of mitochondrial bioenergetics and Ca2+ handling by integrating our recent biophysical models of the CU, NCE, NHE, and CHE into our well-validated model of mitochondrial oxidative phosphorylation, TCA cycle, and electrophysiology. The model also accounts for binding and buffering of cations with metabolites, including ATP, ADP and Pi. Experiments were performed to spectrofluorometrically measure [Ca2+]m, pHm, membrane potential (ΔΨm), and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer medium (ensured with ∼50 μM of EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred with various addition of CaCl2 (0, 10, 25 μM of CaCl2; 16, 88, 130 nM of free [Ca2+] followed by 250 μM of ADP in the presence or absence of carboxyatractyloside (ANT blocker) and oligomycin (F1F0-ATPase blocker). Results and Discussion: Model analysis of the data on (i) initial decrease of [Ca2+]m with addition of Na+-independent substrate HPyr, and (ii) transient increases of [Ca2+]m with addition of ADP suggests ATP/ADP-dependent dynamic Ca2+ buffering inside the cardiac mitochondrial matrix. This model will be helpful to understand mechanisms by which [Ca2+]m both regulates, and is modulated by, mitochondrial energy metabolism.