title: Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A.
abstract: Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.
authors.affiliation: Department of Biomedical Engineering, Institute of Computational Medicine, The Johns Hopkins University, Baltimore MD 21205-2195, USA.
authors.forename: An-Chi
authors.initials: AC
authors.lastname: Wei
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.forename: Sonia
authors.initials: S
authors.lastname: Cortassa
authors.forename: Raimond L
authors.initials: RL
authors.lastname: Winslow
authors.forename: Brian
authors.initials: B
authors.lastname: O'Rourke
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004734:Energy Metabolism; D006168:Guinea Pigs; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010085:Oxidative Phosphorylation; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D019831:Sodium-Calcium Exchanger; C034703:mitochondrial calcium uniporter; D009243:NAD; D016572:Cyclosporine; D002118:Calcium