Myosin heavy chain turnover in cultured neonatal rat heart cells: effects of [Ca2+]i and contractile activity

Abstract

Blockade of L-type Ca2+ channels in spontaneously contracting cultured neonatal rat ventricular myocytes causes contractile arrest, myofibrillar disassembly, and accelerated myofibrillar protein turnover. To determine whether myofibrillar protein turnover. To determine whether myofibrillar atrophy results indirectly from loss of mechanical signals or directly from alterations in intracellular Ca2+ concentration ([Ca2+]i), contractile activity was inhibited with verapamil (10 microM) or 2,3-butanedione monoxime (BDM), and their effects on cell shortening, [Ca2+]i, and myosin heavy chain (MHC) turnover were assessed. Control cells demonstrated spontaneous [Ca2+]i transients (peak amplitude 232 +/- 15 nM, 1-2 Hz) and vigorous contractile activity. Verapamil inhibited shortening by eliminating spontaneous [Ca2+]i transients. Low concentrations of BDM (5.0-7.5 mM) had no effect on basal or peak [Ca2+]i transient amplitude but reduced cell shortening, whereas 10 mM BDM reduced both [Ca2+]i transient amplitude and shortening. Both agents inhibited MHC synthesis, but only verapamil accelerated MHC degradation. Thus MHC half-life does not change in parallel with contractile activity but rather more closely follows changes in [Ca2+]i. [Ca2+]i transients appear critical in maintaining myofibrillar assembly and preventing accelerated MHC proteolysis.

Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments

“…Endothehal cells were pretreated with 20 |^M cytochalasin D for 3 h at 37°C. Staining the cells with FITC-conjugated phalloidin, to visualize F-actin [15,16], showed a significant disruption and degeneration of the organized microfilament network (Fig. 2B) in comparison to untreated cells ( Fig.…”

“…The microarchitecture of the F-actin network before and after cytochalasin D treatment was visualized [15,16] by staining endothelial cells with FITC-conjugated phalloidin (160 nM, 20 min; Molecular Probes). Prior to staining, the cells were fixed (10 min, room temperature) with 2% (w/v) paraformaldehyde in sodium phosphate-buffered saline (PBS), washed (15 min) in 1% (w/v) glycine in PBS, and permeabilized (15 min) with 0.5% (v/v) Triton X-100 in PBS.…”

The circadian protein Clock localizes to the sarcomeric Z-disk and is a sensor of myofilament cross-bridge activity in cardiac myocytes

“…Our results also showed that the presence of Clock in the cytoskeleton was dependent upon active cross-bridge cycling. Both verapamil and BDM decrease the amount of myofilaments present in myocytes but do not significantly reduce non-myofilament proteins like vimentin [21]. This further suggests that the presence of Clock in the cytoskeleton is dependent upon an active contractile apparatus rather than the filamentous cytoskeleton alone.…”

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

“…Previous studies have demonstrated that BDM not only uncouples the E-C coupling of cardiac myocytes, but also inhibits the activity of sarcolemmal L-type Ca 2+ channels (Byron et al 1996;Dooley et al 1999;Ferreira et al 1997). Thus, it was possible that the elongation of intervals of Ca 2+ oscillation was due to the BDM-induced inhibition of the activity of L-type Ca 2+ channels.…”