Myocardial ischaemia is associated with an elevation of cytoplasmic Ca²⁺ ([Ca²⁺]_c) and there is good evidence that disruption of calcium homeostasis plays a role in the damage resulting from ischaemia (McCall et al. 1998; Holmuhamedov et al. 1999). We have used both conventional and confocal fluorescence microscopy to investigate the origin of calcium overload in response to metabolic inhibition with 200 µM 2, 4-dinitrophenol (DNP) of single cardiac myocytes.

Ventricular myocytes were isolated enzymatically from adult rats killed by cervical dislocation, and loaded with fluo-3, Rhod-2, BCECF or tetramethyl rhodamine ethyl ester (TMRE) to assess changes in cytoplasmic or mitochondrial Ca²⁺, pH and mitochondrial membrane potential, respectively. Cells were continuously superfused with normal Tyrode (NT) or Ca²⁺-free solution at 30-32 °C.

Addition of DNP caused rigor after 4-5 min in either NT or Ca²⁺-free solution (cell shortening to 62 ± 4 or 63 ± 3 % of original length, mean ± S.E.M., n = 150). DNP also caused a transient rise in pH_i followed by acidification so that at the time of rigor pH_i was 6.6 ± 0.1 (n = 6). Fluo-3 fluorescence increased in two phases following DNP addition and these increases did not depend on external Ca²⁺. Phase 1 (36 ± 15 % increase, n = 6, P < 0.01 vs. pre-DNP value) occurred immediately and was maintained until rigor. Phase 2 began at rigor and reached 272 ± 3 % after 11 min.

We investigated the origin of Phase 1 using cells loaded with both fluo-3 and Rhod-2. Rhod-2 fluorescence decreased concurrently with the Phase 1 increase in fluo-3 fluorescence. DNP caused immediate mitochondrial depolarisation (measured using TMRE) and the cellular distribution of the dye corresponded to that of Rhod-2. These results suggest that the initial increase in [Ca²⁺]_c on addition of DNP results from release of mitochondrial Ca²⁺.

Phase 2 was abolished when the sarcoplasmic reticulum (SR) was Ca²⁺-depleted with ryanodine, thapsigargin and caffeine prior to DNP addition (P < 0.01 vs. control, n = 6), consistent with this second rise in [Ca²⁺]_c resulting from release of Ca²⁺ from the SR. Statistical comparison was done using ANOVA with multiple comparison to the control using Dunnett’s or an unpaired t test where applicable.

This work was supported by the British Heart Foundation and The Wellcome Trust.