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.