The role of the strong myosin binding site of caldesmon for smooth muscle and cardiac muscle contraction

Phosphorylation of the regulatory light chains of myosin (r-MLC) is believed to be a prerequisite for smooth muscle (SM) contraction. Caldesmon (CaD) is a ubiquitously expressed protein which regulates SM-contraction phosphorylation independently. It contains a N-terminal, myosin binding domain, a central spacer region, and a C-terminal actin, tropomyosin, calmodulin and members of the S100 protein family binding region. Caldesmon and a 20kDa C-terminal actin binding fragment inhibit ATPase activity of phosphorylated myosin and tension in skinned fibers. The function of the myosin binding site is less clear: deletion mutants of the strong myosin binding site were equally effective in inhibiting the actomyosin-ATPase as the WT protein but did not inhibit sliding of actin filaments in an in vitro motility assay. In addition, caldesmon is found in the I-band of the cardiac sarcomere where its function is not known. To elucidate the physiological role of the strong myosin binding site, coded by exon2, we created a mouse line with a deleted exon2 (Delta X2). The Deletion was confirmed by southern blot analysis and sequence analysis of cDNA from either WT or mutant mice. MALDI analysis of recombinant WT and mutant proteins confirmed >45% of the sequence, including specific peptides for the WT protein or the truncated caldesmon. The Delta X2 mice are viable and produce fertile offsprings, with only slightly smaller caldesmon protein. In triton skinned strips from longitudinal ileal SM Ca2+-sensitivity of contraction is not altered while relaxation is slowed down (T-1/2 ileum: Delta X2 86.3s +/- 32.1; WT 56.4 s +/- 11 p<0.05). In isolated cardiac myofibrils (MF) from adult Delta X2 hearts, the slope of the passive length-tension relation is decreased between 2.2 mu m and 2.7 mu m sarcomere length (SL), resulting in a decrease in passive tension at 2.7 mu m SL from 32 +/- 5 (WT) to 17 +/- 3 (Delta X2) nN/mu m(2) (p<0.05). These results suggest, that the strong myosin binding site of caldesmon is involved in the regulation of smooth muscle relaxation and the generation of passive force in the adult heart.