Regulation of myofibrillar accumulation in chick muscle cultures: evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins.

Abstract

The effect of calcium on myofibrillar turnover in primary chick leg skeletal muscle cultures was examined. Addition of the calcium ionophore A23187 at subcontraction threshold levels (0.38 #M) increased significantly rates of efflux of preloaded 45Ca +2 but had no effect on total protein accumulation. However, A23187 as well as ionomycin caused decreased accumulation of the myofibrillar proteins, myosin heavy chain (MHC), myosin light chain If (LClf), 2f (LC2f), a-actin (Ac), and tropomyosin (TM). A23187 increased the degradation rate of LClf, LC2f, and TM after 24 h. In contrast, the calcium ionophore caused decreased degradation of Ac and troponin-C and had no effect on the degradation of MHC, troponin-T, troponin-I, or o~,/~-desmin (Din). In addition, A23187 did not alter degradation of total myotube protein. The ionophore had little or no effect on the synthesis of total myotube proteins, but caused a marked decrease in the synthesis of MHC, LClf, LC2f, Ac, TM, and Dm after 48 h.The mechanisms involved in calcium-stimulated degradation of the myofibrillar proteins were also investigated. Increased proteolysis appeared to involve a lysosomal pathway, since the effect of the Ca ++ ionophore could be blocked by the protease inhibitor leupeptin and the lysosomotropic agents methylamine and chloroquine. The effects of A23187 occur in the presence of serum, a condition in which no lysosomal component of overall protein degradation is detected. The differential effect of A23187 on the degradative rates of the myofibrillar proteins suggests a dynamic structure for the contractile apparatus.

The Effect of Serum Origin on Tissue Engineered Skeletal Muscle Function

“…However, we found that only the TnT isoform correlated with the function of our engineered muscles, whereas both the slow and fast isoforms of TnC and TnI were lower in the USDA tissues. While this was unexpected, the differential expression of the three troponins supports findings that expression of the troponin complex members is not temporally co-ordinated [Silver and Etlinger, 1985;Campione et al, 1993;Brotto et al, 2006;Yu et al, 2007].…”

Segregated assembly of muscle myosin expressed in nonmuscle cells.

“…Similarly, the association of the endogenous MLCs with a MHC fragment expressed in Dictyostelium discoideum does not interfere with the subunit interaction of the endogenous full-length MHC (Manstein et al, 1989). In skeletal muscle, MHCs and MLCs turnover at different rates (Zak et al, 1979;Silver and Etlinger, 1985;Moss et al, 1986); however, there is no consensus on the evidence for free cytoplasmic pools of myosin subunits (Devlin and Emerson, 1978;Zak et al, 1979;Sameral et al, 1987;Gagnon et al, 1989 (Citi et al, 1987;Waschberger and Pepe, 1980). However, in the developing myotube, myosin isotypes are segregated into different cellular structures and compartments.…”

Adult fast myosin pattern and Ca 2+ -induced slow myosin pattern in primary skeletal muscle culture

“…A possible explanation might be a slower degradation of fast MHC isoforms in the presence of ionophore A23187 than of slow MHC I in its absence. Since Silver and Etlinger (32) found no significant effect of A23187, at a concentration identical to ours, on MHC degradation in chicken muscle cultures, a more rapid degradation of slow than of fast MHCs would have to be an intrinsic property of these cells. An alternative explanation is a decreased rate of synthesis of MHCs in the presence of ionophore, and indeed, a decreased rate of synthesis of MHC (of an unspecified type) in the presence of A 23187 has been described by Silver and Etlinger (32).…”