We have investigated the importance of a major component of the cytoskeleton, the microtubules, in the regulation of cardiac cell volume. We used agents to depolymerise (colchicine) or stabilise (taxol) the microtubule proteins, and measured the subsequent changes in myocyte volume during hyposmotic challenge.

Hearts were removed from 11-day-old chick embryos, according to Home Office guidelines, and cells were isolated enzymatically and maintained in culture for 24-72 h. Myocytes were superfused with isosmotic Hepes-buffered saline solution (300 mosmol), and then swollen by exposure to hyposmotic bath solution (170 mosmol, by reduction of [NaCl] ). Myocyte volume was monitored thoughout by capturing digital images of the cells at 1 min intervals. In parallel experiments, cells were fixed in paraformaldehyde and immunolabelled for β-tubulin, to visualise the arrangement of the microtubules. Cells were treated with colchicine (20 µM) or taxol (15 µM) for at least 1 h prior to the experiments, and results were compared with control, untreated cells.

Fluorescence imaging of β-tubulin revealed a distinct arrangement of the microtubules in cardiac myocytes under control conditions; they radiated to the periphery of the cell in all directions from a discrete central point. However, when cells were swollen in hyposmotic solution, this organisation of the microtubules was altered, such that the area around the nucleus became clear and the β-tubulin staining condensed at the cell centre. Most cells showed recovery of the radiating microtubular network on return to isosmotic buffer. β-Tubulin imaging confirmed that taxol treatment prevented this swelling-induced reorganisation of the microtubules, whereas colchicine treatment depolymerised the microtubules even prior to swelling. Pretreatment with either agent completely inhibited the characteristic volume regulatory response (RVD) of these cardiac myocytes, so that cells behaved more as ideal osmometers. When the micro-tubular arrangement was fixed by taxol, hyposmotic challenge caused myocytes to swell to 174 ± 5 % of their volume in control solution (mean ± S.E.M.; n = 4), and volume did not recover significantly in hyposmotic solution. When the microtubules were depolymerised with colchicine, the same challenge caused swelling to 180 ± 4 % of control (mean ± S.E.M.; n = 3), and the cells also remained swollen throughout.

These data show that rearrangement of the microtubular network occurs in cardiac myocytes exposed to a swelling stimulus. This dynamic reorganisation is a necessary component of the regulatory response mediating the subsequent recovery of cardiac cell volume.