Fig. 4.

Bradycardia-induced subcellular secondary Ca²⁺ release (SCR) in systole and Ca²⁺ waves in diastole. A: image of transverse tubules with nonconfocal microscopy; the transverse tubules of striated myocytes from a rabbit heart stained with 4-[β-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) were readily observed using a ×40 objective. B: dual subcellular mapping of APs and CaT from an epicardial myocyte of a Langendorff-perfused rabbit heart stained with PGH-1 and Rhod-2 AM; PGH-1 fluorescence image (top) and optical traces of APs (blue) and CaT (red) from the site marked with an asterisk (top). C: subcellular 150 × 150 μm² field of view seen through Rhod-2 fluorescence. D: CaTs recorded along the line a–a′ identified in C and displayed in a familiar line scan format: CaT during BHR (top), CaT during SHR (middle), and back to CaT during BHR after 5 min SHR (bottom). CaT fluorescence tracings were recorded from a pixel (1.5 × 1.5 μm²) at the center of the line a–a′. Note that SHR leads to low-amplitude Ca_i instabilities and subcellular waves during diastole that are not seen at low magnification and disappear at BHR. Subcellular diastolic Ca_i waves were not synchronized among the neighboring cells. They were averaged out at low magnification. A supplementary movie file is provided to visualize the spatial and temporal heterogeneities of systolic and diastolic Ca_i (supplementary movie 1). E: statistical comparison of the number of secondary Ca²⁺ elevations (SCa_iE; includes both systolic and diastolic elevations) between BHR, SHR, and return back to BHR. The data are derived from all the pixels in the field of view. **P < 0.001.