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First published online July 2, 2004
Journal of Experimental Biology 207, 2755-2767 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01094

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Fiber polymorphism in skeletal muscles of the American lobster, Homarus americanus: continuum between slow-twitch (S₁) and slow-tonic (S₂) fibers

Scott Medler^*, Travis Lilley and Donald L. Mykles

Department of Biology, Colorado State University, Fort Collins, CO 80523, USA

View larger version (115K): [in a new window]   Fig. 1. Alignment of cDNA sequences encoding part of the open reading frame (ORF) and 3' untranslated region (UTR) for the slow-twitch (S1), fast and slow-tonic (S2) myosin heavy chains (MHC) to show sequence similarities and the regions of the S1 and S2 sequences amplified by PCR. Bases that match at least one of the other sequences are shaded, and the stop codons (TAA) are enclosed in boxes. Each sequence shares 79% identity within the ORF. The 3' UTR follows the stop codon and has low sequence identity. S1 forward (S1F), S1 reverse (S1R), S2 forward (S2F) and S2 reverse (S2R) MHC primer positions are indicated by arrows adjacent to the S1 and S2 sequences, respectively. Reverse PCR primers were designed to isoform-specific sequences in the UTR, while forward primers were designed to anneal to regions of relatively low sequence identity in the ORF. The fast and S1 MHC sequences have been published previously (Cotton and Mykles, 1993; Medler and Mykles, 2003). The GenBank accession numbers for the MHC sequences are U03091 (fast), AY232598 (S1) and AY521626 (S2).

View larger version (98K): [in a new window]   Fig. 2. Alignment of cDNA sequences encoding lobster muscle slow-twitch (S1), fast and slow-tonic (S2) tropomyosin (Tm). Bases that match at least one of the other sequences are shaded to indicate the sequence identities among the three isoforms, and the stop codons (TAA) are enclosed in a single box. The beginning of the start codon is indicated. The S2 sequence shares 92% identity with the fast sequence within the ORF, while the S1 and S2 sequences share 97% identity. The S1 and S2 sequences are identical up to bp 833 of the S2 sequence and are then divergent through the rest of the ORF and 3' UTR. A common forward PCR primer was used to amplify both the S1 and S2 sequence, while S1 reverse (S1R) and S2 reverse (S2R) primers were designed to isoform-specific sequences within the 3' UTR. The fast and S1 Tm sequences have been published previously (Mykles et al., 1998). The GenBank accession numbers for the Tm sequences are AF034954 (fast), AF034953 (S1) and AY521627 (S2).

View larger version (43K): [in a new window]   Fig. 3. Alignment of deduced amino acid sequences for the three tropomyosin isoforms (S1, fast and S2). As reported previously (Mykles et al., 1998), the fast and S1 sequences are identical except in the region comprising residues 39-80, resulting in 95% identity. The S2 and S1 isoforms were identical except in five residues at the carboxy-terminal end of the molecule (residues 269-284), resulting in 98% identity between these two proteins.

View larger version (16K): [in a new window]   Fig. 4. Quantification of S2 myosin heavy chain (MHC) mRNA by real-time PCR in different lobster muscle samples (fiber bundles). Expression was highest in the superficial flexors and extensors (SF), followed by the S1 fibers of the crusher claw closer (CR) and then by the fast fibers of the cutter claw closer (CT) and deep abdominal flexors (DA). Differences were analyzed by ANOVA of log-transformed values of copy number per µg of total RNA. A Bonferroni post-hoc test was used to determine which means were significantly different from one another (represented by different lowercase letters). Values are means ± S.D. (N=9-11 samples per group).

View larger version (70K): [in a new window]   Fig. 5. Analysis of S1 and S2 myosin heavy chain (MHC) and tropomyosin (Tm) expression in single fibers from the superficial flexor and extensor muscles by RT-PCR. (A) Individual reactions were run for S1 and S2 MHC primers and then products were combined and separated on the same gel. Plasmids containing either the S1 or S2 MHC cDNA were used as controls. (B) Same analysis as in A, but using the S1 and S2 Tm primers. All of the fibers, with the exception of the second extensor in A, expressed both the S1 and S2 isoforms of MHC and Tm. (Inverse images of ethidium bromide-stained agarose gels.)

View larger version (16K): [in a new window]   Fig. 6. Correlation between the relative proportion of S1 myosin heavy chain (MHC) and S1 tropomyosin (Tm) mRNA in single fibers (semilog scale). The proportion of Tm expressed as the S1 isoform was significantly (P<0.0001; r2=0.698) correlated with the proportion of S1 MHC expressed. The proportion of S1 Tm was always less than that of S2 Tm and no more than 10% of the total Tm expressed, even in fibers classified as S1 fibers. The correlation on a semi-log scale indicates that the relationship between Tm and MHC expression is non-linear (logy=2.35logx-3.84).

View larger version (30K): [in a new window]   Fig. 7. Gradation between S1 and S2 muscle phenotypes identified through SDS-PAGE and western blotting, demonstrating the correlation among MHC, TnT and TnI isoforms. Predominantly S2 muscle samples are on the left (lanes a,b), a predominantly S1 sample is on the right (h), and samples following a gradation from S2 to S1 are in the middle (c-g) (a,b,h are single fibers, while c-g are fiber bundles). Myosin heavy chains (MHC) were identified by SDS-PAGE and silver staining. The S2 MHC isoform has a lower electrophoretic mobility and is identified by a filled circle. The S1 MHC has a higher mobility and is identified by an open circle. The four samples on the left (a-d) only possessed the S2 MHC, the middle samples (e-g) had both S1 and S2 isoforms, and the last sample (h) possessed only the S1 isoform. Troponin (Tn) T and I isoforms were identified by SDS-PAGE followed by western blotting. The TnT isoforms showed an orderly progression from TnT1 (a,b) to both TnT1,3 (c-g) to only TnT3. The TnI isoforms showed a progression from TnI2 (a,b) to TnI2,3 (c-e), to TnI3 (f-h). Various proportions of TnI1 and TnI4 were also observed in several of the samples (d,e,g,h).

View larger version (22K): [in a new window]   Fig. 8. Predominant isoforms in muscle samples (single fibers and bundles of fibers) classified by the myosin heavy chain (MHC) proteins present in the samples (S1, S2 or both MHC proteins). (A) The relative proportion of S1 MHC mRNA was significantly higher in muscles with the S1 MHC protein or both S1 and S2 proteins than in samples possessing only the S2 MHC protein. Unexpectedly, samples characterized by the S2 MHC at the protein level still expressed 30% of their MHC mRNA as the S1 isoform (70% S2 MHC). (B) S1 fibers expressed significantly more troponin (Tn) T as the T3 protein, while mixed samples and those with only the S2 MHC expressed significantly less T3 (more TnT1). (C) TnI2 was the predominant TnI isoform in the S2 fibers, while (D) TnI3 was the major isoform in S1 fibers. Differences were analyzed by ANOVA, and a Bonferroni post-hoc test was used to determine which means were significantly different from one another (represented by different lowercase letters). Values are means ± S.D. [N=21 total samples: S1=10 (single fibers), both=3 (fiber bundles), S2=8 (6 single fibers, 2 fiber bundles)].

View larger version (26K): [in a new window]   Fig. 9. Correlation between TnT and TnI protein isoforms. The proportion of each TnI isoform is plotted as a function of the proportion of TnT3 (1 - proportion TnT1). (A) TnI1, which is expressed in fast muscles (Mykles, 1985a, 1997), was a minor isoform only observed in two samples and was not significantly correlated with TnT composition. (B) The relative amount of TnI2 was negatively correlated with the proportion of TnT3 (P<0.0001; r2=0.79). (C) The relative amount of TnI3 was positively correlated with the proportion of TnT3 (P<0.002; r2=0.58). (D) The proportion of TnI4 was positively correlated with the proportion of TnT3 (P<0.0043; r2=0.41) but was present in lower amounts than TnI3. Arrows at the bottom of the figure indicate that lower amounts of TnT3 (left side of graphs) are characteristic of S2 fibers, while greater amounts of TnT3 (right side of graphs) are characteristic of S1 fibers. Single fiber samples are indicated by filled circles, while samples from small bundles of fibers are represented by open circles.