Cardiac myocytes must maintain their volume within certain limits in order to function effectively and survive. We have investigated the contribution of the mitogen-activated protein kinase (MAPK) signalling cascade to the regulation of cardiac cell volume.

Hearts were removed from 11-day-old chick embryos, according to Home Office guidelines. Myocytes were isolated enzymatically and maintained in culture as single cells (1 day) or confluent monolayers (5-7 days). Cultured myocytes were bathed in isosmotic Hepes-buffered saline solution (300 mosmol), and then swollen by exposure to hyposmotic bath solution (180 mosmol, by reduction of [NaCl]). Myocyte volume was monitored by capturing digital images of single spherical cells at 1 min intervals throughout the experiment. In parallel experiments, proteins were extracted from confluent monolayers of cells by lysis in Triton X-100-containing buffer. Both MAPK protein expression and enzyme activity were measured sequentially by western blotting on the same blot, using specific antibodies; antibody binding was detected by enhanced chemiluminescence and quantified by scanning densitometry.

During hyposmotic challenge, individual cardiac myocytes demonstrated a classical regulatory volume decrease (RVD). Cells swelled initially to 139 ± 11 % of control (mean ± S.D.; n = 11), then recovered significantly to 127 ± 9 % of control after 15 min in hyposmotic solution (P < 0.05; paired t test). MAPK was inactive or barely active in control cells in isosmotic solution (n = 4). Following exposure to hyposmotic solution, significant MAPK activity was detected (P < 0.05, paired t test; n = 4), although protein expression levels remained constant. MAPK was activated transiently, 5-7 min after the onset of the swelling stimulus. Pre-incubating the cells with the specific MEK inhibitor PD98059 (50 µM) for 1 h completely inhibited this swelling-induced increase in MAPK activity (n = 2). Furthermore, this inhibitor completely abolished the RVD response, such that the myocytes remained swollen at 139 ± 8 % of control (mean ± S.D.; n = 5) after 10 min in hyposmotic solution; this volume was significantly greater than the volume of myocytes not treated with the inhibitor, at the same time point (P < 0.05; unpaired t test).

Our data demonstrate that transient activation of MAPK occurs rapidly following the onset of cell swelling. This stimulated MAPK activity appears to be an essential component of the signalling cascade that mediates volume regulation following cardiac cell swelling.