The impact of MyBP-C family members on sarcomeric MyHC organization

Abstract

Striated muscle consists of a complex array of proteins, the precise alignment and organization of which are essential for proper muscle function. Many of the proteins that comprise the sarcomere, the basic unit of striated muscle tissue, have been assigned to one of three different filamentous systems: the elastic, thin and thick filament systems. Two proteins that are associated with the thick filament are myosin binding protein (MyBP)-C and MyBP-H. Within the sarcomere, these proteins are found in a series of transverse stripes within the A-band. The function of these MyBPs remains to be determined. Both MyBPs are capable of altering the ATPase activity of myosin, and ultrastructural analyses demonstrate that they can regulate the diameter of myosin filaments formed in vitro. These data imply a role for MyBPs both in sarcomere organization and muscle contraction.;Previous analyses of the interactions among sarcomeric proteins have relied on biochemical techniques. In this thesis, cultured non-muscle cells were transfected with cDNAs encoding sarcomeric proteins to analyze the interaction between MyBPs and myosin heavy chain (MyHC), and to identify the regions of these proteins which mediate this interaction. Cultured cardiac myocytes were used to investigate the impact of full-length and mutant MyBPs on sarcomeric architecture. I show that: (1) MyBPs dramatically modulate the organization of MyHC expressed in non-muscle cells. (2) The modulation of MyHC organization by MyBP-H is mediated through the 24 carboxy-terminal amino acids of MyBP-H. (3) MyBPs regulate the diameter of MyHC containing structures. (4) Full-length MyBPs are incorporated into the sarcomeres of cardiac myocytes. Deletions at the amino terminus of MyBP-C do not effect this incorporation, while deletions at the carboxyl terminus of both MyBPs abolish it. (5) Both MyBPs bind to MyHC between amino acids 1193 and 1629. (6) Binding of MyBPs to myosin fragments does not cause a change in myosin distribution as does binding of MyBPs to full-length MyHC. Analysis of these data leads to a model to describe the role of MyBPs in thick filament organization and sarcomere architecture.