title: Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling.
abstract: Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.
authors.affiliation: Hatter Institute for Cardiology Research, MRC Inter-University Cape Heart Group, University of Cape Town Medical School, Cape Town, South Africa.
authors.forename: J
authors.initials: J
authors.lastname: Minners
authors.forename: L
authors.initials: L
authors.lastname: Lacerda
authors.forename: J
authors.initials: J
authors.lastname: McCarthy
authors.forename: J J
authors.initials: JJ
authors.lastname: Meiring
authors.forename: D M
authors.initials: DM
authors.lastname: Yellon
authors.forename: M N
authors.initials: MN
authors.lastname: Sack
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006880:Hydroxy Acids / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D019157:Ischemic Preconditioning, Myocardial; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D003652:Decanoic Acids; D006880:Hydroxy Acids; D014665:Vasodilator Agents; C052853:5-hydroxydecanoic acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D000241:Adenosine; D003981:Diazoxide