Formamide-induced osmotic shock has previously been used to detubulate isolated adult rat ventricular myocytes (i.e. disrupt the surface membrane-t-tubule junction; Kawai et al. 1999); these studies suggested that the major Ca²⁺ influx and efflux pathways – I_Ca and I_Na/Ca, respectively – are concentrated in the t-tubules. In the present study, confocal microscopy was used to investigate detubulation-induced changes in Ca²⁺ influx and efflux. Rats were killed by Schedule 1 procedure. Isolated ventricular myocytes were exposed to formamide for 15 min as described previously (Kawai et al. 1999). Formamide was then washed out for 30 min with Tyrode solution, before loading the cells with fluo-3 AM (to monitor cytosolic [Ca²⁺]). Figure 1 shows transverse linescan images of a fluo-3-loaded normal myocyte (right) and formamide-treated myocyte (left) during electrical stimulation. In normal cells, the rise of [Ca²⁺] was relatively uniform across the cell width.However, in formamide-treated cells, a localised increase in [Ca²⁺] initially occurred close to the surface membrane, and then propagated towards the centre of the cell. This supports the suggestion that the t-tubules play an important role in Ca²⁺ influx, allowing a temporally and spatially homogeneous increase of intracellular Ca²⁺. A different protocol was used to study the effects of detubulation on Ca²⁺ extrusion. This involved rapidly decreasing the bathing [Ca²⁺] to ~100 nM, followed by introduction of 10 µM fluo-3 in the bathing solution. In normal cells, application of 20 mM caffeine resulted in a rapid rise in [Ca²⁺] outside the cell. However, in formamide-treated cells, the efflux of Ca²⁺ was much less pronounced, consistent with the suggestion that the Na⁺-Ca²⁺ exchanger is concentrated in the t-tubules.This work was supported by the British Heart Foundation and The Wellcome Trust.

Figure 1. Transverse linescan images of a normal myocyte (right) and a formamide-treated myocyte (left) during electrical stimulation. Bathing solution contained (mM): 113 NaCl, 5 KCl, 1 MgSO4, 1.0 CaCl2, 1.0 Na2HPO4, 20.0 sodium acetate, 10.0 glucose and 10.0 Hepes. Room temperature.

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