Ca2+ channel regulation by transforming growth factor-beta 1 and bone morphogenetic protein-2 in developing mice...

In skeletal muscle myogenesis, precursor cells or myoblasts fuse to form multinucleated cells (myotubes), which then further develop into functional muscle. We investigated if the inhibition of myogenesis by transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) involve regulation of voltage-dependent Ca(2+) channels. Primary cultured myoblasts were kept in fusion medium (0-6 days) in either the absence (control conditions) or the presence of 40 pm TGF-beta1 or 5 nm BMP-2. Subsequently, the developing myotubes were transferred to a growth factor-free recording solution, and subjected to whole cell patch-clamp experiments. At day 0, 14% of non-fusing myoblasts exhibited T-current, whereas the L-current was practically absent. Under control conditions, however, the percentage of T- and L-channel-expressing myotubes increased sharply, from 25% at day 1 to approximately 100% at days 2-6. In addition, parallel increases were determined for Ca(2+)-currents density and cell membrane capacitance (C(m)), which is proportional to the size of myotubes. Interestingly, at days 1-2 TGF-beta1 and BMP-2 eliminated the T-current on initial 14% of T-channel-expressing myoblasts. Moreover, at day 6 the growth factors significantly reduced the maximal values of both T-current density (80%) and C(m) (60%). The effect of BMP-2 was selective on T-channels, whereas TGF-beta1 decreased also the L-current density (90%). A similar reduction in maximal conductance of the Ca(2+) channels was determined, in the absence of significant alterations in other essential properties of the channels, including the time course and voltage dependence of activation and inactivation. The results suggest these growth factors markedly reduce the number of functional T- (both TGF-beta1 and BMP-2) and L-channels (only TGF-beta1) in the surface of the plasma membrane, and contribute to explaining the associated effects on myogenesis.