Muscular performance varies enormously among animals, as well as even within a single organism, and some of the variation is speculated to arise from adaptations of troponin, which regulates tension development in striated muscle. Like vertebrates, C. elegans has multiple genes coding for troponin I (TnI), the inhibitory subunit within the ternary troponin complex. Phylogenetic analysis of the four C. elegans TnIs suggested that diversification of TnI isoforms occurred independently in vertebrates and invertebrates. Nonetheless, the domain architecture of TnI is preserved in all isoforms. RNA interference (RNAi) of TnI-1 (F42E11.4), expressed in embryonic body wall muscle and hermaphroditic gonad, led to no discernible defect. Similar results were obtained following RNAi of TnI-4 (W03F8.1), expressed in the pharyngeal muscle. RNAi of the larval and adult body wall muscle TnI, Unc-27/TnI-2 (ZK721.2), produced rigid paralysis mimicking that of
unc-27 mutants. Elimination of TnI-3 (T20B3.2) yielded constipated and egg-laying defective worms, consistent with detection of TnI-3 mRNA in vulval muscles and intestine. Three mutant alleles of
unc-27 exist:
e155 (Gln10stop), a presumed null;
su142 sd (Gln122stop), which lacks the domain thought necessary for inhibition at sub-micromolar Ca 2+ ; and
su195 sd (Glu207stop), which eliminates only the C -terminus. Each of the mutant alleles severely compromised swimming behavior of adult homozygous hermaphrodites (p<0.0001), and among the mutants,
su142 sd worms showed the lowest tail-beat frequency (p<0.001). Even at the L1 stage,
su142 sd worms differed from wild type in swimming (p<0.05). Both polarized light microscopy and anti-vinculin labeling of mutant body-wall muscle revealed a loss of sarcomeric organization. The wild-type pattern of obliquely arranged, alternating isotropic and anisotropic bands was absent. Dense bodies, marking the ends of the sarcomeres, were evenly spaced along parallel rows in wild-type worms, but their disposition was greatly distorted in the mutants. Electron microscopy of body wall muscle confirmed a prevalent disorder of dense body positioning and a less well defined sarcomere structure. The reason for this appearance is identified from transverse sections. In wild-type worms this orientation showed a regular alternation of the sarcomere bands. Dense bodies were followed by I bands, composed of thin filaments, then by overlap zones of A bands, with regularly packed thick filaments surrounded by rings of thin filaments, and next by the H zone of A bands, containing only thick filaments. In the mutants, the same bands were seen, but they were not in a regular pattern. Small islands of thin filaments were interspersed within the overlap region of A bands and even within the H zone. In the overlap region the content of thin filaments was quite variable, and the H zone was fragmented into several small areas of the cross section. The defects were strongest in the
su142 sd mutants. This structural pattern could arise from unregulated contraction of the sarcomere, in which small portions of each myofibril shorten irregularly and independently from one another, thereby distorting the filament disposition. Collectively, the functional and structural analyses are consistent with a primary role of TnI in inhibiting force development and highlight the dependence of sarcomeric organization on proper contractile activity. The exacerbated deficits exhibited by
su142 sd worms strengthen the view that the N -terminal portion of TnI participates in enhancing force development in an active muscle and thus suggest that design variations within this portion of TnI isoforms could contribute to the diversity of tension-producing capability of muscle.