Confocal microscopy was used to study nuclear Ca²⁺ regulation in permeabilized cells or in intact myocytes during electrical stimulation. Rats (250-300 g) were killed humanely (Schedule 1) and ventricular myocytes were isolated by enzymatic digestion. Following permeabilization with saponin (10 µg/ml), myocytes were perfused with a mock ‘intracellular’ solution containing (mM) 100 KCl; 25 HEPES; 0.36 EGTA; 10 phosphocreatine; 5 ATP and fluo-3 acid (5 µM). The free [Ca²⁺] and [Mg²⁺] was 200 nM and 1 mM respectively. pH 7.0, 22 ^oC. Myocytes typically had two elongated nuclei oriented along the mid axis of the cell. Line scan (x-t) images were obtained by positioning the scan line longitudinally through both nuclei. Under these conditions, prolonged Ca²⁺ release events were consistently detected from the nuclei. The mean duration, amplitude and width of the spontaneous NCR events was 1.78 ± 0.19 s, F/Fo = 2.0 ± 0.04 and

6.1 ± 0.2 µm respectively (n=68). For comparison, the mean half time, amplitude and width of spontaneous Ca²⁺ sparks under the same conditions was 34.3 ± 1.1 ms, F/Fo =2.0 ± 0.1 and 2.6 ± 0.12 µm respectively (n=43). The location of spontaneous nuclear Ca²⁺ release (NCR) events was identified more accurately using syto11 (250 nM), which fluoresces on binding nucleic acids, allowing the boundary of the nucleoplasm and the NE to be defined. NCR was shown to occur in localised regions at the ends of the elongated nuclei and in-focus events were aligned with the inner surface of the nuclear envelope (NE). Ca²⁺ release events of comparable duration were not detected when the scan-line was positioned transversely across the midpoint of each nucleus or from cytosolic regions, which were clearly outside the nucleus. In further experiments, intact myocytes were perfused with a solution containing mM: 113 NaCl; 5.4 KCl; 1 MgCl₂; 1.0 CaCl₂; 0.37 Na₂HPO₄; 5.5 glucose; 5 HEPES. 20-22^oC. Cells were then loaded with fluo-3 AM (5 µM for 10 minutes) to allow changes in intracellular [Ca²⁺] to be detected during field stimulation. Each rise in cytosolic [Ca²⁺] was followed an increase in nuclear [Ca²⁺]. However, line-scan and x-y images revealed regions at the ends of the nuclei, where [Ca²⁺] increased rapidly and reached a higher level than the maximum cytosolic [Ca²⁺], before diffusing inwards towards the centre of the nucleus. The inherently prolonged nature of the spontaneous NCR events detected at the ends of the nucleus in skinned cells suggests the presence of Ca²⁺ release sites with markedly different gating properties to those involved in the generation of cytosolic Ca²⁺ sparks. The experiments on intact cells suggest that synchronised recruitment of these events may serve to amplify and prolong the rise in nuclear [Ca²⁺], which accompanies each systolic Ca²⁺ transient.