Ischemic and Pharmacological Preconditioning in Girardi Cells and C2C12 Myotubes

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 (DCm) 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 DCm (JC-1: simulated ischemia 9063%, adenosine 8267%, diazoxide 8764%, versus control 100%, P,0.05); (2) attenuation in cellular ATP levels (CTL 0.2160.03 nmol/L ATP/mg protein, simulated ischemia 0.1260.02, adenosine 0.1560.02, diazoxide 0.1160.02, P,0.05); and (3) enhanced cellular oxygen consumption (control 2.360.1 nmol/L oxygen/min/1310 cells, simulated ischemia 3.160.1, adenosine 3.160.3, diazoxide 2.660.2, 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. (Circ Res. 2001;89:787-792.)