Regulation of striated muscle contraction

Abstract: Background and Aims: Muscle contraction involves cross-bridge interaction between actin and myosin filaments, which is regulated by variations of intracellular [Ca2+] and influenced by several external control systems. To characterize the mechanism of contraction, we have studied the effects of pharmacological compounds influencing the actin-myosin interaction and/or Ca2+ activation systems. We examined the effects of blebbistatin, a novel inhibitor of the actomyosin ATPase, in the organized contractile system of muscle (papers I and II). Cardiac contraction can be influenced by extracellular factors and in paper III, we examined the inotropic action of UTP and UDP, which can be released under ischemic conditions. The recent developments in molecular biology and the genetic characterization of several muscle diseases have created a need to link specific genes and proteins to functions in muscle. The zebrafish (Danio rerio) has become a useful vertebrate model organism in this context. The main aim of paper IV was to develop techniques for studies of skeletal muscle in the zebrafish larvae and to characterize its mechanical and structural properties. Results and Conclusions: In paper I, blebbistatin inhibited contraction of the cardiac preparations of the mouse with the half-maximal inhibitory concentration in the micromolar range. Using permeabilized cardiac preparations, we showed that blebbistatin did not influence Ca2+-sensitivity of the contractile filaments. Using patch clamp technique, we showed that blebbistatin neither interfered with action potential duration nor Ca2+ influx. Thus, in mouse heart, blebbistatin directly inhibits actin myosin interactions. In Paper II, we extended the observation of blebbistatin's ability to bind differently to several isolated myosin isoforms in vitro: the contractions in a range of skeletal, cardiac and smooth muscle tissues, all showing different myosin isoform profiles, were differently inhibited by blebbistatin. Thus, blebbistatin's selectivity for different myosin isoforms is also present in the organized contractile system. Using permeabilized skeletal muscle where active force measurements were combined with small angle x-ray diffraction (synchrotron sourced), we further showed that blebbistatin interacted with a nucleotide-bound myosin state and trapped the myosin cross-bridge in a configuration dissociated from actin. In paper III, we examined extracellular control mechanisms for cardiac contraction focusing on pyrimidines which are released in the human during ischemia. They act on isolated cardiac muscle in a receptor-specific way through an IP3-pathway which behaves independently from cAMP. Thus, we established that pyrimidines are an external control mechanism for cardiac shortening acting independently of the usual inotropic mechanisms. In paper IV, we developed a novel method to study structure and mechanical properties of skeletal muscle of zebrafish in the larval stadium. Single twitch and tetanus contractions could be recorded at an optimal sarcomere length of 2.15 ?m. Clear equatorial x-ray diffraction patterns could be recorded showing that the myofilaments were mainly arranged in parallel to the long axis of the larval preparation. A smooth tetanus was observed at a high stimulation frequency. Repeated tetanic stimuli uncovered that fatigue developed rapidly in these muscles. These results show that the zebrafish larvae muscle have a fast muscle phenotype. Permeabilized muscle preparations showed a lower Ca2+- sensitivy of contraction compared with mouse skeletal muscle. Thus, zebrafish larval skeletal muscle displays a fast contractile profile where a high cytoplasmic Ca2+- concentration for contraction is combined with rapidly developing fatigue. The study of the influence of different levels of muscle gene expression on the mechanical behavior of the tissue has with these experiments become a distinct possibility.