Key Points
- To investigate how the loss of transverse tubules during short-term culture alters spatial calcium influx and sarcoplasmic reticulum calcium release in adult ventricular myocytes.
- Simultaneously recorded intracellular calcium transients and membrane currents in freshly isolated (1-4 hours) and short-term cultured (1-2 days) guinea-pig ventricular myocytes using confocal microscopy and whole-cell patch clamping.
- Dialyzed cells intracellularly with the low-affinity calcium buffer citrate to distinguish calcium influx from sarcoplasmic reticulum release.
- Assessed t-tubule structure and sarcolemmal integrity using membrane capacitance measurements and Di-8-ANEPPS fluorescence staining.
- Freshly isolated myocytes exhibited spatially uniform calcium entry and release, whereas short-term cultured myocytes developed marked spatial inhomogeneities and calcium waves propagating from cell ends.
- Cultured myocytes showed a decrease in total cell capacitance to 65% accompanied by a loss of functional t-tubular membrane connected to the surface.
- Spatial gradients in calcium influx directly triggered restricted, non-uniform calcium release from the sarcoplasmic reticulum in cells lacking t-tubules.
Structured PICO
PPopulationGuinea-pig ventricular myocytes
IInterventionShort-term culture (1-2 days)
CComparatorFreshly isolated adult myocytes (used within 1-4 h of isolation)
OOutcomeSpatial properties of intracellular Ca2+ transients and membrane currentssurrogate
The t-tubular network is functionally important for uniform ventricular Ca2+ signaling, as its loss in short-term culture leads to spatially non-uniform SR Ca2+ release.