Evolutionary significance of myosin heavy chain heterogeneity in birds.

This article reviews the complexity, expression, genetics, regulation, function, and evolution of the avian myosin heavy chain (MyHC). The majority of pertinent studies thus far published have focussed on domestic chicken and, to a much lesser extent, Japanese quail. Where possible, information available about wild species has also been incorporated into this review. While studies of additional species might modify current interpretations, existing data suggest that some fundamental properties of myosin proteins and genes in birds are unique among higher vertebrates. We compare the characteristics of myosins in birds to those of mammals, and discuss potential molecular mechanisms and evolutionary forces that may explain how avian MyHCs acquired these properties.

Repression of myosin isoforms in developing and denervated skeletal muscle fibers originates near motor endplates.

During development of chicken pectoralis muscle, a neonatal myosin heavy-chain isoform is supplanted progressively by an adult isoform. This expression is under neuronal control. In this study we test the hypothesis that developmental myosin transformations are initiated near the motor endplate of each muscle fiber, thereafter progressing toward the fiber ends. By using immunocytochemical methods, pectoralis muscle from chickens aged 1-115 days after hatching were labeled by antibody against neonatal isoform. Ellipse minor axis and mean optical density of labeled and/or unlabeled fiber profiles from each bird were measured by computer image analysis. Acetylcholinesterase (AChE) activity was demonstrated histochemically. Using serial cross sections, we show that smaller fiber profiles are the tapered ends of larger fiber profiles. The largest fiber profiles (central regions of the fibers) were the first to lose their neonatal myosin during development. Motor endplates were localized by AChE activity to the central regions of the fibers. The pectoralis of mature chickens was denervated for 3, 7, 15, or 21 days. After 2 weeks' denervation, neonatal myosin is first reexpressed in the fiber ends. Dev Dyn 2000;217:50-61.

Persistent expression of developmental myosin heavy chain isoforms in the tapered ends of adult pigeon pectoralis muscle fibres.

We have shown previously that in addition to the adult myosin heavy chain (MyHC) isoform present throughout the length of each fast-twitch glycolytic muscle fibre within the pectoralis of the mature chicken, the neonatal isoform is retained in the tapered ends of these fibres. This work, however, has been the only published report of this phenomenon. Here, we tested the hypothesis that similar to the chicken, the ends of mature pigeon pectoralis muscle fibres contain developmental MyHC isoform(s). A histological stain was used to visualize endomysium to assist in the analysis of transverse sections of pectoralis muscle from four mature pigeons. Immunocytochemical techniques were used to localize MyHC isoform(s) characteristic of pigeon pectoralis development. We show that within mature pigeon pectoralis, the ends of both fast-twitch glycolytic and fast-twitch oxidative-glycolytic fibre types express MyHC isoform(s) characteristic of their earlier development. Thus, we extend our findings on chicken to another species and an additional muscle fibre type. Retention of developmental MyHC isoform(s) within the tapered ends of mature muscle fibres may be more widespread than is currently appreciated.

Functional properties of myosin isoforms in avian muscle.

Sarcomeric myosin is the major skeletal muscle protein and is encoded by a large and complex multigene family whose members are differentially expressed in developing and adult muscle cells. The structure and function of sarcomeric myosins have been extensively analyzed and many myosin genes have now been cloned and sequenced. This manuscript reviews the broad spectrum of myosin research with emphasis on studies in avian systems and discusses how advances in myosin isoform analysis have contributed to muscle and meat science.

Regulation of alpha-helical coiled-coil dimerization in chicken skeletal muscle light meromyosin.

The dimerization specificity of the light meromyosin (LMM) domain of chicken neonatal and adult myosin isoforms was analyzed by metal chelation chromatography. Our results show that neonatal and adult LMMs associate preferentially, although not exclusively, as homodimeric coiled-coils. Using chimeric LMM constructs combining neonatal and adult sequences, we observed that a stretch of 183 amino acids of sequence identity at the N terminus of the LMM was sufficient to allow the adult LMM to dimerize in a non-selective manner. In contrast, sequence identity in the remaining C-terminal 465 amino acids had only a modest effect on the dimerization selectivity of the adult isoform. Sequence identity at the N terminus also promoted dimerization of the neonatal LMM to a greater degree than sequence identity at the C terminus. However, the N terminus had only a partial effect on the dimerization specificity of the neonatal sequence, and residues distributed throughout the LMM were capable of affecting dimerization selectivity of this isoform. These results indicated that dimerization preference of the neonatal and adult isoforms was affected to a different extent by sequence identity at a given region of the LMM.

Expression of myosin heavy chain isoforms during development of domestic pigeon pectoralis muscle.

The pectoralis muscle of birds provides virtually all the power for the downstroke of the wing during flight. In adults it consists almost entirely of FOG (fast-twitch oxidative-glycolytic) and/or FG (fast-twitch glycolytic) fiber types. The aims of this study are to contrast MyHC (myosin heavy chain) transitions occurring within avian FG and FOG fibers during development, and to test the hypothesis that the pectoralis matures before the acquisition of flight. Pectoralis was obtained from pigeons (Columba livia) aged from 13 days in ovo to adult. Monoclonal antibodies generated against chicken MyHC isoforms were used with Western blots and immunocytochemistry. FG and FOG fibers were differentiated using a histochemical method demonstrating NADH (nicotinamide adenine dinucleotide), and "lesser fiber diameters" were quantified. Western blots confirm that the antibodies label pigeon MyHCs. A small number of the fibers are slow type in ovo, but these are quickly restricted in distribution and lost after hatching. In ovo fast-twitch fibers contain a ventricular isoform, and at least two embryonic-neonatal forms (designated E-N103 and E-N165). One week after hatching, fast-twitch fibers can be distinguished by NADH as FG or FOG. At fledging, four weeks after hatching, FG and FOG fibers are smaller than in older birds and E-N103 and E-N165 persist in both fibertypes. E-N103 wanes in all fibers shortly after fledging. E-N165 gradually disappears from FG fibers. Thus, despite pigeons being at adult body mass at fledging, their pectoralis is not fully mature.

The role of interhelical ionic interactions in myosin rod assembly.

Interhelical electrostatic interactions at specific heptad positions can regulate dimerization specificity of alpha-helical coiled-coils. We have analyzed 20 vertebrate myosin sequences from a variety of organisms and tissues in order to determine if interhelical ionic interactions correlate with the observed myosin dimerization specificity. We find that the sites for potential interhelical ion pairing are identical in virtually all sarcomeric myosins whether they form homo- or heterodimers. We also show that smooth muscle and non-muscle myosin rod sequences exhibit a different conserved pattern of potential interhelical ion pairing. These observations suggest that myosin rod residues involved in interhelical electrostatic interactions do not regulate dimerization specificity, but may contribute to the specific arrangements of myosin molecules that determine differences in the filament morphology of sarcomeric and non-sarcomeric muscles.

Cloning, nucleotide sequence and characterization of a full-length cDNA encoding the myosin heavy chain from adult chicken pectoralis major muscle.

Four cDNA clones, encoding the chicken adult sarcomeric MyHC, have been isolated from a pectoralis major muscle cDNA library using gene-specific DNA probes. These clones were sequenced and then subcloned into a full-length, 6-kb, chicken adult sarcomeric MyHC cDNA. The entire cDNA consists of 5873 nucleotides with 19 bp 5'-untranslated region and 34 bp 3'-untranslated region. The complete cDNA encodes a 1939-aa polypeptide whose molecular weight is 223 kDa. The calculated isoelectric point of this protein is approximately 5.7. Analysis of the deduced amino acid sequence and comparison with a previously published amino-acid sequence of the same MyHC isoform reveals that six amino acid residues are different. Hydrophilicity analysis of this adult MyHC amino-acid sequence shows a similar pattern as the embryonic MyHC. A recombinant baculovirus, carrying this full-length adult MyHC cDNA, has also been generated and expressed in the Sf9 insect cell line. A approximately 220-kDa recombinant MyHC was synthesized and reacted specifically with chicken adult MyHC monoclonal antibodies.

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken.

Two unique cDNA clones containing chicken slow myosin heavy chain (MyHC) inserts have been isolated from an expression library. Immunochemical analyses of the expressed proteins using different slow MyHC specific monoclonal antibodies were consistent with the two clones encoding slow MyHC 1 (SM1) and slow MyHC 2 (SM2) protein sequences. Northern blot analyses showed that the clones hybridized with 6-kb mRNAs that are differentially expressed in developing and adult slow muscles, further supporting the conclusion that these two clones represent SM1 and SM2 cDNAs. Sequence analyses show that both clones encode the highly conserved light meromyosin portion of the sarcomeric myosin rod and are 78-81% homologous to a mammalian slow/cardiac beta-MyHC cDNA. Hybridization using PCR generated probes specific for SM1 and SM2 sequences demonstrated that the genes encoding these two slow MyHCs colocalized to an 80-kb BssHII genomic fragment. We further show that a probe specific to a third slow MyHC gene also hybridized with the same 80-kb genomic fragment. We conclude that in the chicken genome there is a slow MyHC locus containing at least three distinct slow MyHC genes.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle.

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

Innervation regulates myosin heavy chain isoform expression in developing skeletal muscle fibers.

The influence of innervation on primary and secondary myogenesis and its relation to fiber type diversity were investigated in two specific wing muscles of quail embryo, the posterior (PLD) and anterior latissimus dorsi (ALD). In the adult, these muscles are composed almost exclusively of pure populations of fast and slow fibers, respectively. When slow ALD and fast PLD muscles developed in ovo in an aneurogenic environment induced after neural tube ablation, the cardiac ventricular myosin heavy chain (MHC) isoform was not expressed. The adult slow MHC isoform, SM2, appeared by embryonic day 7 (ED 7) in normal innervated slow ALD but was not expressed in denervated muscle. Analysis of in vitro differentiation of myoblasts from fast PLD and slow ALD muscles isolated from ED 7 control and neuralectomized quail embryos showed no fundamental differences in the pattern of MHC isoform expression. Newly differentiated fibers accumulated cardiac ventricular, embryonic fast, slow SM1 and SM3 MHC isoforms. Nevertheless, the expression of slow SM2 isoform in myotubes formed from slow ALD myoblasts only occurred when myoblasts were cultured in the presence of embryonic spinal cord. Our studies demonstrate that the neural tube influences primary as well as secondary myotube differentiation in avian forelimb and facilitates the expression of different MHC, particularly slow SM2 MHC gene expression in slow myoblasts.

Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle.

The aims of this study are to investigate the diversity of myosin heavy-chain (MyHC) expression among avian fast-twitch fibers, and to test the hypothesis that dissimilar MyHC isoforms are found in each of the principal avian fast-twitch fiber types. MyHCs within the muscle fibers of the pectoralis of 31 species of bird are characterized using immunocytochemical methods. A library of 11 monoclonal antibodies previously produced against chicken MyHCs is used. The specificity of these antibodies for MyHCs in each of the muscles studied is confirmed by Western blots. The results show that avian fast-twitch glycolytic fibers and fast-twitch oxidative-glycolytic fibers can contain different MyHCs. Among the species studied, there is also a conspicuous variety of MyHC isoforms expressed. In addition, the results suggest that two epitopes are restricted to chickens and closely allied gallinaceous birds. There are no apparent correlations between between MyHC epitope and presupposed contractile properties. However, the presence of different isoforms in different fast-twitch fiber types suggests a correlation between isoform and contractile function.

Myosin heavy chain expression within the tapered ends of skeletal muscle fibers.

BACKGROUND: The pectoralis muscle of the chicken contains fast-twitch glycolytic fibers, which during development undergo a transformation in their myosin heavy chain (MyHC) content from embryonic to a neonatal to an adult isoform (Bandman et al., 1990). Little, however, is known of MyHC expression within the ends of these or other muscle fibers. Here we test the hypothesis that the tapered ends of mature skeletal muscle fibers contain a less mature MyHC isoform than that typically found throughout their lengths. METHODS: We apply an ammoniacal silver histological stain for endomysium and monoclonal antibodies against neonatal and adult MyHCs of chicken pectoralis to transverse serial sections of pectoralis from five mature chickens. The "lesser fiber diameters" of populations of fibers from each bird are also measured. RESULTS: Most (approximately 81.8%) of the small (< 12 microns) and none of the larger (> 20 microns) diameter fibers contain the neonatal MyHC. Following these smaller fibers through serial sections, we show that they are the tapered ends of the larger fibers. Whereas neonatal MyHC is restricted to the tapered fiber ends, adult MyHC is present throughout the entire lengths of all fibers. We also demonstrate acetylcholinesterase (AChE) activity at some of these fiber ends. CONCLUSIONS: We postulate that longitudinal growth of myofibrils in adult muscle is characterized by the sequential expression of MyHC isoforms similar to that observed in rapidly growing muscle and that the presence of the neurotransmitter hydrolase AChE at the tapered fiber ends may be related to the retention of neonatal MyHC.

Tough calls: making ethical decisions in the care of older patients.

Medicine is a moral endeavor concerned with the good of treating those who are ill. An ethical decision is the justifiable response to a "should" question, with consequences of good or harm. Three conditions are necessary for a patient to share in making an ethical decision: competence, voluntariness, and knowledge related to the condition of illness. In cases of incapacity, either the standard of substitute judgment or of best interest may be applied. Ideally, an ethical decision is a shared decision between providers and patients. In cases of conflict, continuing communication and dialogue are morally superior to the use of power.

The evolution of the chicken sarcomeric myosin heavy chain multigene family.

This manuscript describes the chicken sarcomeric myosin heavy chain (MyHC) multigene family and how it differs from the sarcomeric MyHC multigene families of other vertebrates. Data is discussed that suggests the chicken fast MyHC multigene family has undergone recent expansion subsequent to the divergence of avians and mammals, and has been subjected to multiple gene conversion-like events. Similar to human and rodent MyHC multigene families, the chicken multigene family contains sarcomeric MyHC genes that are differentially regulated in developing embryonic, fetal, and neonatal muscles. However, unlike mammalian genes, chicken fast MyHC genes expressed in developing muscles are also expressed in mature muscle fibers as well. The potential significance of conserved and divergent sequences with the MyHC rod domain of five fast chicken isoforms that have been cloned and sequenced is also discussed.

The evolutionary relationship of avian and mammalian myosin heavy-chain genes.

Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat alpha- and beta-cardiac myosin heavy-chain sequences suggest that gene-conversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.

Contractile protein isoforms in muscle development.

The contractile proteins of skeletal muscle are often represented by families of very similar isoforms. Protein isoforms can result from the differential expression of multigene families or from multiple transcripts from a single gene via alternative splicing. In many cases the regulatory mechanisms that determine the accumulation of specific isoforms via alternative splicing or differential gene expression are being unraveled. However, the functional significance of expressing different proteins during muscle development remains a key issue that has not been resolved. It is widely believed that distinct isoforms within a family are uniquely adapted to muscles with different physiological properties, since separate isoform families are often coordinately regulated within functionally distinct muscle fiber types. It is also possible that different isoforms are functionally indistinguishable and represent an inherent genetic redundancy among critically important muscle proteins. The goal of this review is to assess the evidence that muscle proteins which exist as different isoforms in developing and mature skeletal and cardiac muscles are functionally unique. Since regulation of both transcription and alternative splicing within multigene families may also be an important factor determining the accumulation of specific protein isoforms, evidence that genetic regulation rather than protein coding information provides the functional basis of isoform diversity is also examined.

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.