Functional Impact of Cell Culture on Excitation-Contraction Coupling in Canine Myocytes

Abstract

Downregulation of ion currents as well as a loss of T-tubules is documented during heart failure. A similar reduction of both has been observed in cultured ventricular myocytes. We further assessed the functional impact of cell-culture on excitation-contraction (EC) coupling in dog ventricular, atrial and Purkinje myocytes. Ventricular, atrial and Purkinje myocytes were isolated and cultured for up to 48 hours. Myocytes were stained with di-8-ANEPPS to visualize ultrastructure and Ca2+ transients (CaTs) were recorded by confocal microscopy. Ion channel currents were assessed via patch electrode voltage clamp. Membrane staining with di-8-ANEPPS indicated dog ventricular myocytes having an extensive T-tubular network; atrial myocytes having a rudimentary T-tubule system at best, and Purkinje myocytes having no T-tubules. Transverse X-t lines scans of electrically stimulated ventricular myocytes showed CaTs rising synchronously across the cell, whereas in Purkinje myocytes the CaT rise showed a U-shaped profile. CaT rise in atrial myocytes typically showed a U-shaped profile, but less pronounced than that in Purkinje myocytes. Ventricular myocytes cultured for 48 hours indicated a dramatic loss of T-tubules and a switch from a synchronous CaT rise to a U-shaped profile. In cultured atrial cells, no loss of T-tubules nor alterations in CaTs was noted. ICa in ventricular myocytes cultured for 48 hours was reduced by 21%. Comparative assessment of culture-induced changes in ion channel currents in atrial myocytes is ongoing. While cultured ventricular myocytes show a loss of T-tubules and phenotypic switch from a homogeneous CaT rise to a ‘U’-shaped profile, atrial myocytes cultured identically show little change in ultrastructure and CaT profile. These results suggest that character and degree of change in EC coupling induced by similar pathological insults markedly depends on cardiac myocyte type.