Published online before print September 13, 2001, doi: 10.1161/hh2101.098372
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© 2001 American Heart Association, Inc.
Cellular Biology |
Ischemic and Pharmacological Preconditioning in Girardi Cells and C2C12 Myotubes Induce Mitochondrial Uncoupling
From the Hatter Institute for Cardiology Research, MRC Inter-University Cape Heart Group (J.M., L.L., J.McC., J.J.M., M.N.S.), University of Cape Town Medical School, Cape Town, South Africa; and the Hatter Institute for Cardiovascular Studies (D.M.Y.), University College Hospitals and Medical School, London, United Kingdom.
Correspondence to Michael N. Sack, MD, PhD, Hatter Institute for Cardiology Research, UCT Medical School, Observatory 7925, Cape Town, South Africa. E-mail sack{at}capeheart.uct.ac.za
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 (
m) 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 m (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/µg 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/1x106 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 KATP-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.
Key Words: preconditioning • mitochondrial function • mitochondrial membrane potential • ATP • oxygen consumption
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