The ability of mitochondria to take up cytosolic calcium ([Ca²⁺]_c) may affect overall cellular activity by modulating the amplitude and duration of Ca²⁺ signals as well as mitochondrial ATP production via an elevation of mitochondrial matrix [Ca²⁺]. The principal driving force for mitochondrial ATP production is the mitochondrial membrane potential (Δσ_m), which may be reduced by mitochondrial Ca²⁺ uptake. The present communication sought to relate changes in [Ca²⁺]_c to changes in Δσ_m. To examine this, Δσ_m was measured using the membrane potential-sensitive dye tetramethylrhodamine ethyl ester (TMRE) simultaneously with fluo-4 to monitor [Ca²⁺]_c during sarcolemma-depolarisation- and inositol (1,4,5)P₃ (InsP₃)-evoked [Ca²⁺]_c increases in single voltage clamped myocytes (from guinea-pigs, ~500 g, humanely killed by stunning and exsanguination).

Treatment with either carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which uncouples oxidative phosphorylation, plus oligomycin to inhibit mitochondrial ATP hydrolysis or with the Complex I inhibitor rotenone plus oligomycin depolarised mitochondria, as assessed by the loss of punctate TMRE fluorescence. Mitochondrial depolarisation increased the time required for [Ca²⁺]_c to recover to resting values following either sarcolemma-depolarisation-induced Ca²⁺ influx or release of Ca²⁺ from the intracellular store by flash photolysis of caged InsP₃, indicating that mitochondrial Ca²⁺ accumulation occurred during these [Ca²⁺]_c transients. Despite this no alteration of TMRE fluorescence and hence Δσ_m was observed during the [Ca²⁺]_c transients. Infrequently the fluorescence of a single mitochondrion decreased, as if partially or completely depolarised, but the timing of these events often occurred after [Ca²⁺]_c had returned to resting levels. Oligomycin alone blocks proton re-entry into the mitochondrial matrix, causing a small Δσ_m hyperpolarisation. This was observed as an increased mitochondrial fluorescence, without significant alteration to either depolarisation- or InsP₃-evoked Ca²⁺ transients. The muscarinic agonist carbachol transiently increased [Ca²⁺]_c with no detectable alteration in Δσ_m. On occasion this increase was followed by [Ca²⁺]_c oscillations and waves. During the course of these prolonged Ca²⁺ oscillations individual mitochondria could become either partially or completely depolarised, but the timing of this occurrence was not correlated with [Ca²⁺]_c or the Δσ_m of surrounding mitochondria.

In conclusion, mitochondria may be functionally independent entities and under the conditions of the present experiment increases in [Ca²⁺]_c do not have an immediate effect on mitochondrial membrane potential.

This work was supported by The Wellcome Trust and BHF.