Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multi-step immunofluorescent analysis of vitamin D receptor loci and myosin heavy chain isoforms in human skeletal muscle

Vitamin D receptors have been shown to be present in human skeletal muscle using different techniques. We developed a multi-staining immunofluorescent method to detect vitamin D receptor expression and co-localize it with myosin heavy chain isoform expression in skeletal muscle biopsies in older female subjects. Serial sections were cut from frozen samples obtained by needle biopsy of the vastus lateralis. Samples were probed with a primary vitamin D receptor monoclonal antibody and then re-probed with a type IIa myosin heavy chain isoform-specific antibody. Independent unfixed sections followed a similar protocol and were probed with type IIx and type I myosin heavy chain isoform-specific antibodies. Immunohistochemistry and fluorescent microscopy co-localized vitamin D receptor loci and myosin heavy chain isoforms in whole skeletal muscle sections. We quantified intranuclear vitamin D receptor staining patterns and number of individual muscle fiber subtypes within a muscle section. Immunohistochemical staining of the vitamin D receptor was confirmed by Western blot using the same monoclonal antibody. This multi-staining immunofluorescent technique allows for measurement of intranuclear vitamin D receptor expression in the context of the specific muscle fiber type profile in a single section. This method can thus be a useful approach to study potential relationships between muscle fiber subtypes and vitamin D receptor expression.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.     

Differing ADP release rates from myosin heavy chain isoforms define the shortening velocity of skeletal muscle fibers

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).     

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5′-regions of the three known genes encoding light meromyosin isoforms (10 °C, intermediate and 30 °C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 °C and intermediate types, between the 10 °C and 30 °C types, and between the intermediate and 30 °C types, respectively. Isoform-specific differences were clearly observed between the 10 °C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 °C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 °C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.     

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.     

Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures.

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.     

Smooth muscle-type myosin heavy chain isoforms in bovine smooth muscle and non-muscle tissues

Summary— The distribution of smooth muscle (SM)-type myosin heavy chain isoforms in several bovine muscular and non-muscular (NM) tissues was evaluated by immunofluorescence tests using monoclonal antibodies SM-E7, reactive with 204 (SM1) and 200 (SM2) kDa isoforms, and SM-F11, specific for SM2 isoform. SM-E7 reacted equally with vascular, respiratory and intestinal SM tissues, whereas SM-F11 stained heterogeneously SM cells in the various muscular systems examined and in some peculiar tissues was unreactive (perisinusoidal cells of hepatic lobule, pulmonary interstitial cells and intestinal muscularis mucosae) or uniquely reactive (nerve cells). On the whole, our findings indicate that SM1 and SM2 isoforms are unequally distributed at the cellular level in various SM and NM tissues and support previous results obtained with tissue extracts and electrophoretic procedures.     

The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle

Hypertrophy was produced in the anterior latissimus dorsi (ALD) muscle of 5-wk-old chickens by application of a load to the humerus. After 4 wk, hypertrophied ALD muscles were greater than 2.5 times heavier than contralateral control ALD muscles. Two isomyosins are distinguishable in normal ALD muscles by their different electrophoretic mobilities. It is shown here that the faster migrating SM-1 isomyosin decreases in abundance with age and that the application of an overload enhances both the rate and extent of this process. Monoclonal antibodies were selected by an immunotransfer technique that were specific for the heavy chains associated with either SM-1 or SM-2, or cross-reacted with both isoforms. The cellular distribution of the SM-1 and SM-2 isomyosins was analyzed by immunofluorescent technique using these antibodies. Anti-SM-1 and anti-SM-2 antibodies reacted with separate populations of cells, whereas the third antibody reacted with all myocytes in the normal ALD muscle. These data suggest that there is an exclusive cellular distribution of myosin heavy chains associated with SM-1 and SM-2 proteins. Immunofluorescent analysis of hypertrophied muscle showed the anti-SM-2-specific antibody reacting with all myocytes, whereas the anti-SM-1-specific antibody reacted with none. This is consistent with the elimination of the SM-1 isoform in hypertrophied muscles.     

Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.     

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav_1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav_1.5 positive fibers (1548±70 μm²) were 35% smaller than Nav_1.5 negative fibers (2367±78 μm²; P<0.05) in SEN muscle, whereas Nav_1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm²; P<0.05) where no Nav_1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav_1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.     

Expression of myosin heavy chain isoforms in myogenic clones obtained from developing quail breast muscle

Summary Monoclonal antibodies specifically recognizing cardiac and extraocular muscle myosin heavy chains of the quail (Coturnix coturnix japonica) were used to determine the patterns of expression of these isoforms in clonal cultures of embryonic quail myoblasts. Myoblasts prepared from 9 day embryonic pectoralis are virtually homogeneous in their ability to form clones expressing both cardiac and extraocular isoforms. The majority of myoblasts obtained from day 5 embryos also formed clones which co-express the cardiac and extraocular isoforms, but a small percentage of the clones expressed only cardiac isoforms.     

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Dimerization specificity of adult and neonatal chicken skeletal muscle myosin heavy chain rods

The dimerization specificity of the recombinantly expressed and purified rod domain of adult and neonatal chicken myosin heavy chain was analyzed using metal chelation chromatography. Our results indicate that full-length adult and neonatal rods preferentially formed homodimers when renatured from an equimolar mixture of the two isoforms denatured in guanidine hydrochloride. The contribution made toward the dimerization specificity by subdomains of the rod has been addressed by making a chimeric protein consisting of the subfragment 2 (S2) region of the adult isoform and the light meromyosin region of the neonatal isoform. The proportion of heterodimers formed in exchange experiments between the chimera and the neonatal and adult rods rose with increase in the sequence homology between the two exchanging proteins. This suggests that multiple regions of the rod domain of chicken MyHC including S2 can contribute toward dimerization specificity.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.