The rate of loss of T‐tubules in cultured adult ventricular myocytes is species dependent

Abstract

In this study, we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72 h culture period. The T-tubule density was measured in the following two ways: (i) as whole-cell capacitance in voltage-clamped myocytes relative to cell area; and (ii) using di-8-ANEPPS staining and confocal microscopy. In adult rat ventricular myocytes, whole-cell capacitance/area was significantly reduced from 47 +/- 3 fF microm(2) (mean +/- s.e.m.; n = 16) in freshly isolated (control) cells to 36 +/- 2 fF microm(2) (n = 20) after 72 h in culture. The T-tubular density, as assessed optically using di-8-ANEPPS staining, at 48 h was significantly reduced to 70 +/- 7% (n = 14) compared with control cells. The T-tubular density was further reduced after 72 h in culture to 43 +/- 7% (n = 10) compared with control cells. In contrast, in mouse myocytes neither whole-cell capacitance relative to cell area nor optical assessment of T-tubules showed any significant reduction in capacitance/cell area or T-tubule density after 72 h of culture. Expression of caveolin-3 (CAV-3) (a marker of T-tubule development) was also measured, and a significant reduction was observed in CAV-3 expression in rat myocytes at 48 (80 +/- 5.5%; n = 6) and 72 h (66 +/- 9.5%; n = 6) compared with control cells. The expression of CAV-3 in mouse myocytes was not significantly reduced even at 72 h. When rat ventricular myocytes were paced in culture for 72 h they exhibited no significant improvement in T-tubule density or CAV-3 expression compared with non-paced cultured cells. In rat myocytes, sarcomere length shortening was significantly reduced in myocytes cultured for 48 (4.96 +/- 0.72%; n = 26) and 72 h (4.32 +/- 0.80%; n = 26) compared with freshly isolated cells (7.12 +/- 0.56%; n = 18). Mouse myocytes, after 24 h in culture, were unable to follow external pacing. These results suggest that detubulation in quiescent culture is slower in the mouse than the rat and that this loss of T-tubules profoundly affects excitation-contraction coupling in rat myocytes.