Isolation of newly synthesised myosin filaments from skeletal muscle homogenates and myofibrils

Abstract

LARGE and rapid changes in the concentrations of myofibrillar protein can occur during hypertrophy and atrophy of skeletal and cardiac muscle^1,2. Understanding of the regulation of these changes will require analysis of the steps between the initiation of transcription and the degradation of the protein to amino acids. The role of post-translational steps in these processes has generally been ignored since there has been no way to isolate biochemically intermediates of myofibrillar assembly. This is unfortunate since the physiological and metabolic regulation of such steps may control the rate of incorporation of new protein into this organelle, and thus the levels of functional contractile protein in the cell. Present understanding of myofibrillar assembly has come mainly from ultrastructural studies. In embryonic³, regenerating⁴ and rapidly growing muscles from young animals⁵, electron microscopy suggests that polymerised thick and thin filaments can aggregate to form myofibrils de novo and/or insert into pre-existing myofibrils. In contrast, microscopy of normal adult muscle usually does not reveal these structural aspects of myofibrillogenesis, although isotope studies showed that contractile proteins are constantly synthesised and degraded in the muscle cell^1,2,6, even in the absence of net growth. This may be because only a small fraction of protein exists as nascent filaments not yet integrated into fibrillar sarcomeres, and/or because these assembly steps may be too rapid to be captured by electron microscopy. We report here the isolation of a population of thick and thin myofilaments that may constitute such an intermediate in myofibrillar assembly.