Spatially non‐uniform Ca2+ signals induced by the reduction of transverse tubules in citrate‐loaded guinea‐pig ventricular myocytes in culture.

1. Ratiometric confocal microscopy and the whole‐cell patch clamp technique were used to simultaneously record intracellular Ca2+ transients and membrane currents from guinea‐pig ventricular myocytes. Intracellular dialysis with the low‐affinity Ca2+ buffer citrate enabled us to record and analyse Ca2+ transients caused by Ca2+ influx alone and by additional Ca2+ release from the sarcoplasmic reticulum (SR) in the same cell. 2. In freshly isolated adult myocytes (used within 1‐4 h of isolation) both types of Ca2+ transients (‘Ca2+ entry’ and ‘Ca2+ release’ transients) were spatially uniform regardless of the Ca2+ current (ICa) duration. In contrast, Ca2+ transients in short‐term cultured (1‐2 days) myocytes exhibited marked spatial inhomogeneities. ICa frequently evoked Ca2+ waves that propagated from either or both ends of the cardiac myocyte. Reduction of the ICa duration caused Ca2+ release that was restricted to one of the two halves of the cell. 3. Analysis of the Ca2+ entry signals in freshly isolated and short‐term cultured myocytes indicated that the spatial properties of the Ca2+ influx signal were responsible for the spatial properties of the triggered Ca2+ release from the SR. In freshly isolated ventricular myocytes Ca2+ influx was homogeneous while in short‐term cultured cells pronounced Ca2+ gradients could be found during Ca2+ influx. Spatial non‐uniformities in the amplitude of local Ca2+ entry transients were likely to cause subcellularly restricted Ca2+ release. 4. The changes in the spatial properties of depolarization‐induced Cai2+ signals during short‐term culture were paralleled by a decrease (to 65%) in the total cell capacitance. In addition, staining the sarcolemma with the membrane‐selective dye Di‐8‐ANEPPS revealed that, in cultured myocytes, t‐tubular membrane connected functionally to the surface membrane was reduced or absent. 5. These results demonstrate that the short‐term culture of adult ventricular myocytes results in the concomitant loss of functionally connected t‐tubular membrane. The lack of the t‐tubular system subsequently caused spatially non‐uniform SR Ca2+ release. Evidence is presented to show that in ventricular myocytes lacking t‐tubules non‐uniform SR Ca2+ release was, most probably, the result of inhomogeneous Ca2+ entry during ICa. These findings directly demonstrate the functional importance of the t‐tubular network for uniform ventricular Ca2+ signalling.