Development of connectin/titin and nebulin in striated muscles of chicken

Abstract

The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca2+ transients. To investigate the relationship between Ca2+ transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca2+ transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca2+ transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca2+ oscillations, but a sustained increase in intracellular Ca2+ (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca2+ oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca2+ oscillatory signal may be decoded through reorganization of the integrin–cytoskeletal protein complex via calpain-mediated proteolysis.