Formamide has been used previously to detubulate isolated rat cardiac ventricular myocytes (Kawai et al. 1999). The purpose of the current investigation was to determine what happens to the t-tubules during this procedure. Wistar rats were killed humanely using a Schedule 1 method. Ventricular myocytes were enzymatically isolated, and detubulated using formamide as described by Kawai et al. (1999).

Control ventricular myocytes in which the cell membrane was stained with the lipophilic dye di-8-ANNEPS clearly showed an extensive t-tubular system (Fig. 1A; 31/31 cells). Cells stained after treatment with formamide showed only surface staining up to 1 h (Fig. 1B; 28/34 cells) and at 5 h (6/6 cells) after treatment, indicating detubulation. Cells stained with di-8-ANNEPS and then treated with formamide showed a different pattern (Fig. 1C; 34/39 cells): clear transverse (t-tubular) striation was absent, although staining was still visible within the cell.To investigate whether vacuoles are formed by the disrupted t-tubules, as in skeletal muscle (Krolenko et al. 1998), cells were incubated for 2 h with 100 µg ml^-1 fluorescein-dextran (MW 10 000), which is unable to enter the cell, followed by formamide treatment in dextran-containing solution. Subsequent washout of formamide in dextran-free solution resulted in no fluorescence signal visible in the cell (10/10 cells). However, following washout of formamide with solution containing dextran, localised fluorescence was clearly visible within the cell (12/15 cells). Cell size did not change after detubulation (2681.9 ± 106.8 µm² in control (n = 53) vs 2632.6 ± 71.1 µm² in detubulated cells (n = 63; mean ± S.E.M.; P > 0.05, Student’s t test).

These data suggest that detubulation occurs on removal of formamide and persists for at least 5 h. The t-tubules appear to be present, possibly as vacuoles, within the cell following detubulation, although not apparently connected to the surface sarcolemma. It appears unlikely that the previously reported formamide-induced decrease in cell capacitance (Kawai et al. 1999) is due to a decrease in cell size, but is more likely due to physical, and hence electrical, uncoupling of the t-tubules from the surface membrane.This work was supported by the British Heart Foundation and The Wellcome Trust.

Figure 1. Confocal images of representative ventricular myocytes stained with di-8-ANNEPS. A, control cell. B, cell stained after formamide treatment. C, cell stained and then treated with formamide. Horizontal white bar, 50 µm.

Kawai, M., Hussain, M. & Orchard, C.H. (1999). Am. J. Physiol. 277, H603-609.

Krolenko, S.A., Amos, W.B., Brown, S.C., Tarunina, M.V.& Lucy, J.A. (1998). J. Mus. Res. Cell Mot. 19, 603-611.