Muscles in the squid, Doryteuthis pealeii, can extend further than muscles in vertebrate animals. Thompson et al. (2015) have demonstrated that the amount of force a muscle maintains as it stretches, referred to as the length-force relationship (LFR), differs significantly between squid muscle types. The mechanisms responsible for these differences are not understood, but it is likely that length-force relationships (LFRs) are regulated by large muscle proteins. Twitchin is known to play a role in controlling muscle activity in other organisms. The gene for twitchin was first identified in C. elegans by Benian et al. (1993) when mutations within it resulted in muscle twitching. Butler and Seigman (2010) have suggested that twitchin may function by establishing connections between filaments in smooth muscles that enable the maintenance of force. Distinct forms (isoforms) of twitchin may enable muscles types to express divergent contractile properties. In mussels, Funabara et al. (2005) have shown that twitchin isoforms play a role in the “catch” state, where muscle contraction is maintained using minimal energy. Our hypothesis, that squid muscles with distinct LFRs express different twitchin isoforms, was investigated by comparing sequences of RNA expressed by muscle fibers with distinct LFRs. Since RNA sequence determines the structure of proteins like twitchin, differential expression of twitchin RNA that correlates with LFR characteristics would support our hypothesis. Our results indicate that distinct isoforms of twitchin do exist in the squid funnel retractor and head retractor muscles and that alternative processing of RNA from the twitchin gene is responsible for these isoforms. This discovery provides insight into mechanisms regulating smooth muscle contraction.
