Functional Role of T-tubules on Calcium Transients in Canine Cardiac Myocytes

Abstract

Background: A loss of t-tubules and alterations in ultrastructure occur in cultured ventricular myocytes. A similar alteration in t-tubules and ultrastructure is well documented under certain pathological conditions such as heart failure. We examined the ultrastructural changes in cultured canine cardiac cells and determined the functional impact these changes have on excitation-contraction coupling and Ca2+ transients. Materials, Methods and Results: Atrial, ventricle, and Purkinje myocytes were isolated by enzymatic dispersion. Atrial and ventricular myocytes were cultured for up to 48 h. Voltage clamp recordings were made with patch electrodes. Ca2+ transient was recorded as a laser scanning confocal microscope in myocytes loaded with Ca2+ fluorescent dyes. Membrane ultrastructure was imaged in myocytes stained with the membrane selective dye, di-8-ANEPP. Freshly isolated ventricular myocytes had a well-developed t-tubule system, while Purkinje cells had no t-tubules; some atrial cells exhibited a primitive t-tubule system. In atrial and Purkinje cells, the Ca2+ transient had a U-shaped profile with the fluorescence highest at the edge of the cell. In contrast, ventricular myocytes showed a homogeneous rise in Ca2+ at the edge and center of cells. Ventricular myocytes cultured for 2 days showed a nearly complete loss of t-tubules. The Ca2+ transients revealed a phenotypic switch from a homogeneous profile to a “U”-shaped profile. Interestingly, atrial cells in culture maintained their primitive t-tubule system. Conclusions: Our results show that atrial and ventricular myocytes respond differently to being placed in culture. Ventricular myocytes, but not atrial myocytes, quickly lose their t-tubules accompanied by a Ca2+ transient profile suggestive of a phenotypic switch in Ca2+ handling. The differential response also suggests that the various cardiac tissue types would respond differently to pathophysiological stresses.