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First published online September 9, 2003

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Analysis of myofibrillar proteins and transcripts in adult skeletal muscles of the American lobster Homarus americanus: variable expression of myosins, actin and troponins in fast, slow-twitch and slow-tonic fibres

Scott Medler and Donald L. Mykles^*

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

View larger version (42K): [in a new window]   Fig. 1. Diagram of cutter claw with lateral portion of exoskeleton removed to reveal the arrangement of the closer muscle. The central region of the muscle is composed of large fibers that are the fast phenotype. The ventral and distal regions of the closer are slow, with the ventral region composed of S1 fibers and the distal region composed of S2 fibers. Previous work by Lang et al. (1980) demonstrated that these regions are controlled by phasic (central), tonic (distal) or both (ventral) motor neurons. The same study showed that the distal region of both claws possessed highly oxidative fibers. The basic anatomy of the crusher claw is similar to that of the cutter but is composed entirely of S1 fibers.

View larger version (92K): [in a new window]   Fig. 2. Alignment of cDNA sequences encoding part of the open reading frame (ORF) and 3' untranslated region (UTR) for fast and slow (S1) MHCs to show the regions amplified by PCR. Bases that match one another are shaded, and the stop codon is enclosed in a box. The S1 MHC sequence and the fast MHC sequence share 79% identity within the ORF. The 3' UTR follows the stop codon and has lower sequence identity. Fast forward (FF), fast reverse (FR), slow forward (SF) and slow reverse (SR) MHC primer positions are indicated by arrows adjacent to the fast and slow sequences, respectively. Reverse PCR primers were designed to anneal to specific sequences in the 3' UTR, while forward primers were designed to anneal to regions of relatively low sequence identity in the ORF. The fast MHC sequence has been published previously (Cotton and Mykles, 1993). The GenBank accession numbers for the fast and S1 MHC sequences are U03091 and AY232598, respectively.

View larger version (36K): [in a new window]   Fig. 3. Correspondence between myosin heavy chain (MHC) protein composition and other myofibrillar isoforms identified through SDS-PAGE and western blotting. (A) 8% silver-stained gel demonstrating different MHC isoforms present in various muscles. MHC isoforms could not be distinguished between the fast [deep abdominal flexor (DA) and central cutter (CCT)] and S1 [ventral cutter (VCT) and crusher (CR)] muscles. However, the S2 fibers [distal cutter (DCT) and superficial tail flexors (SF)] contained an additional isoform that migrated more slowly than the fast or S1 isoforms (arrowheads mark positions of the isoforms). Some slow muscles contained a mixture of the S1 and S2 MHC isoforms (DCT), while others contained only the S2 isoform (SF). The double band at the far left is rabbit MHC used as a control (Sigma). (B) 10% silver-stained gel showing other myofibrillar isoforms from the samples identified in A. Some of the dominant bands correspond to MHC, paramyosin isoforms P1 and P2, P75, troponin T (TnT), actin (A), tropomyosin (Tm) and troponin I (TnI). Fast fibers characteristically possess paramyosin isoforms P1 and P2, P75, TnT2, TnI1, TnI3 and TnI5. S1 fibers possess P2, TnT3, TnI2 and TnI4. S2 fibers are characterized by P2, TnT1, TnT3, TnI1 and TnI2. (C) Composite of three western blots of the above samples probed with antibodies to P75, TnT and TnI. Most of the above isoform differences are observed when the blot is labeled with these antibodies.

View larger version (21K): [in a new window]   Fig. 4. Correlation between S1 and S2 myosin heavy chain (MHC) isoforms and troponin T1 (TnT1) found in slow fibers from the distal region of the cutter claw. (A) Representative fibers demonstrate that some fibers contain only the S2 MHC isoform (a,b), some fibers contain both the S1 and S2 isoforms (c,d) and some fibers contain only the S1 isoform (e,f). Rabbit MHC (Sigma) is the double band to the left of lane a. (B) Western blot probed with anti-TnT from the fibers shown in A. In general, the fibers containing larger amounts of the S2 MHC isoform also contain a large proportion of TnT1. Exceptions to this trend are observed in the fiber in lane f, which possesses only the S1 MHC isoform but also contains TnT1, and the fiber in lane d, which contains the S2 MHC isoform but only has the TnT3 isoform. (C) Correlation between the percentage of TnT existing as the T1 isoform and the percentage of the S2 MHC isoform found in different slow fibers. A significant correlation exists between TnT1 and the S2 isoform [% TnT1=1.56+0.29(% S2), r2=0.59, P<0.0009]. While a correlation exists between these two markers for S2 fibers, the relationship is not absolute and accounts for only 60% of the variability in the proportion of TnT1 in the fibers. These data support the notion that S1 and S2 fibers exist in a continuum between pure S1 and S2 fibers. It is also apparent that the TnT1 isoform is never present by itself but, at most, accounts for about 50% of the TnT protein in a fiber.

View larger version (55K): [in a new window]   Fig. 5. Alignment of deduced amino acid sequences for major overlapping portions of the ORFs of the fast and S1 myosin heavy chain (MHC) sequences (amino acids that match exactly are shaded). The corresponding base pairs are indicated on the left. The deduced sequences from the fast and S1 ORFs share 81% identity and 91% similarity in this region of the molecule. The S1 and fast forward MHC PCR primers begin at positions 1193 bp and 938 bp, respectively.

View larger version (60K): [in a new window]   Fig. 6. Analysis of myosin heavy chain (MHC), actin and P75 expression in fast fibers by RT-PCR. (A) Individual reactions were run for each primer set (fast MHC, S1 MHC or P75) and then products were combined and separated on the same gel. Plasmids containing target sequences (fast MHC, S1 MHC or P75) were used as controls. (B) Separate reactions were run for actin and separated on a second gel. Deep abdominal flexor muscle (lane a) expressed fast MHC and P75, but actin expression was not detected. Other samples from these muscles expressed actin but at low levels compared with other fibers. Three individual fast fibers from the central region of the cutter claw (lanes b, c and d) expressed both fast and S1 MHC, P75 and actin. (Inverse images of ethidium bromide-stained gels.)

View larger version (60K): [in a new window]   Fig. 7. Analysis of myosin heavy chain (MHC), actin and P75 expression in slow fibers by RT-PCR. Experimental arrangement is as in Fig. 6. Different slow fibers [distal cutter (lane a), ventral cutter (lane b), crusher (lane c), lateral superficial extensor (lane d)] predominantly expressed S1 MHC and actin. The distal cutter (lane a) shows low amounts of fast MHC product. Actin expression (B) with this method appears to be higher than S1 MHC expression (A). (Inverse images of ethidium bromide-stained gels.)

View larger version (15K): [in a new window]   Fig. 8. Quantification of myosin heavy chain (MHC) and actin mRNAs by real-time PCR. (A) Deep abdominal muscles exclusively expressed fast MHC and significantly lower levels of actin. (B) Fast cutter claw muscles predominantly expressed fast MHC, but variable amounts of S1 MHC were also detected. Actin expression was lower but not significantly different from the levels of fast MHC expression. (C) Crusher muscles expressed significantly more S1 MHC than fast MHC. Actin expression was significantly higher than MHC expression in the slow muscles. Differences were analyzed by ANOVA of log-transformed values of copy number per mg of tissue. A Bonferroni post-hoc test was used to determine which means were different from one another (represented by different uppercase letters). Values are means ± S.D. (N=10 samples).

View larger version (21K): [in a new window]   Fig. 9. Correlation between S1 myosin heavy chain (MHC) expression and actin expression. A significant correlation was observed between S1 MHC expression and actin expression: S1 MHC expression=0.437+0.716(actin expression), r2=0.537, P<0.0001. This correlation was dominated by the differences between the fast cutter fibers (CT) and the S1 fibers. When these groups were analyzed separately, a significant correlation was still observed for the cutter fibers (r2=0.643, P<0.009) but not for the S1 fibers from the crusher claw muscles (r2=0.027, P<0.52).