Nederlandse Organisatie voor Wetenschappelijk Onderzoek - NWO
The contractile unit of skeletal muscle is the so-called sarcomere. The text book view of the sarcomere consists of two types of myofilaments (actin and myosin based) that slide past each other and that thereby cause muscle contraction. However, two additional myofilaments were discovered recently, i.e. titin and nebulin. Whereas the role of titin is beginning to be understood, the in vivo functions of the giant protein nebulin are largely unknown. So far several hypotheses have been proposed that are based on experiments with highly simplified in vitro systems. I recently observed reduced nebulin expression in diaphragm muscle of patients with chronic obstructive pulmonary disease. This finding directed me towards the study proposed here. To conclusively establish the role of nebulin requires study of the intact myofilament system. The tools of choice for such studies are the nebulin knock-out mouse and the nebulin exon microarray, tools recently developed by Professor Granzier and colleagues. The proposed research will be conducted in Professor Granzier?s laboratory. Firstly, I will elucidate whether nebulin participates in regulating the interaction between myosin and actin. In particular, I will investigate the role of nebulin in regulating cross-bridge binding. This work includes measurement of the force-pCa relation, ATPase activity, and cross-bridge cycling kinetics in skinned fibers from wild-type and nebulin KO mice. Secondly, I will study whether nebulin determines the actin filament length, thereby regulating its overlap with myosin. It is a widely held belief that nebulin is a 'molecular ruler' that determines the length of the actin filament, but direct evidence is nonexistent. Hence, I will measure thin filament length and nebulin exon composition in skeletal muscle of wild-type and nebulin KO mice, as well as determine their force-sarcomere length relations (a non-structural measure of actin filament length).