Localization of sarcomeric proteins during muscle assembly in mouse cardiomyocytes and skeletal myotubes

Abstract

This study seeks to investigate the role of contractions in myofibrillogenesis, and structure of nascent myofibrils. The model system employed in these experiments was cultured quail myotubes. In order to determine the role of contractions in myofibrillogensis, contractions were indirectly inhibited with elevated KCl, and directly inhibited with 2,3 butanedione monoxime (BDM), a small cell-permeable inhibitor of actin & muscle myosin interactions. Myotubes were treated with contraction inhibitors at 2½days –soon after the main fusion event. On the 6thculture day, there was significant delay in myofibrillogensis in myotubes exposed to elevated KCl. This delay was characterized by the expansion of nascent myofibrils at the spreading edges of myotubes. This delay in myofibrillogenesis was not accompanied by diminished accumulations of muscle myosin. On the 4thculture day, there was complete arrest of myofibrillogenesis at the nascent step with the treatment of BDM. As a result, it is concluded that contractions are necessary for the progression of nascent myofibrils to mature myofibrils. The structure of nascent myofibrils was further investigated with super resolution microscopy. Structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy revealed mini-A-bands –shorter than 1.5 μmin length –associated with nascent myofibrils. These structures were spaced at varying intervals, and oriented at angles deviating the from the actin superstructure of the nascent myofibril. Mini-A-bands were also observed to progressively in expand in length distal to the spreading edges of myotubes. STED imaging indicates that some mini-A-band are adjacent to, and not integrated within, the actin superstructure of nascent myofibrils.