Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling

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 (Delta Psim) 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 (greater than or equal to 30%, P <0.05 versus ischemic controls) and a reduction in propidium iodide uptake by greater than or equal to 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 Delta Psim (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/mug 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.