Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S1) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

Cloning and tissue expression of eleven troponin-C isoforms in the American lobster, Homarus americanus.

Troponin-C is the Ca(2+)-binding subunit of the troponin regulatory complex in striated muscles. As TnC isoforms can influence the Ca(2+)-activation properties of fiber phenotypes, the diversity and tissue distribution of TnC cDNAs were assessed in the American lobster, Homarus americanus. We cloned ten full-length cDNAs and one partial cDNA coding for distinct TnC isoforms. Five were sequenced from expressed sequence tag clones and were designated Ha-TnC(2b)(') (2094 nt, 141 aa and 15.9 kDa), -C(4)(') (1667 nt, 155 aa and 17.3 kDa), -C(5) (2884 nt, 149 aa and 17.3 kDa), -C(6) (2439 nt, 155 nt and 17.4 kDa) and-C(6x) (2171 nt, 154 aa and 16.9 kDa). The remainder were cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends: five full-length cDNAs, designated Ha-TnC(1) (814 nt, 150 aa and 17.1 kDa), -C(2a) (639 nt, 152 aa and 17.2 kDa), -C(2b)('') (2136 nt, 155 aa and 17.5 kDa), -C(3) (1046 nt, 150 aa and 16.9 kDa), -C(4)('') (842 nt, 108 aa and 12.1 kDa) and one partial (3') cDNA, designated Ha-TnC(4)(''') (563 nt and 57 aa). Ha-TnC(1), -C(2a), and-C(2b)(') corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC(1), -C(2a), and-C(2b)). Alternative splicing appeared responsible for TnC(2b)(') and-C(2b)(''); TnC(4)('), -C(4)('') and-C(4)('''); and TnC(6) and-C(6x). The deduced amino acid sequences differed primarily in the terminal regions and EF-hands I and III. Ha-TnC(6x) had a highly divergent 76 aa proline-rich N-terminal sequence. Tissue expression of the Ha-TnC isoforms was analyzed qualitatively by endpoint PCR. Ha-TnC(1), -C(2a), -C(2b)('), -C(2b)('') and-C(3) were expressed primarily in skeletal muscles; Ha-TnC(5) was expressed in heart; and Ha-TnC(4) and-C(6) variants were expressed in muscles and other tissues. The number and diversity of TnC sequences suggest the potential for varying the Ca(2+)-activated properties of the troponin-tropomyosin regulatory complex through differential expression of TnC isoforms.