Everett Bandman

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.

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.

Myosin Isoenzyme Transitions in Muscle Development, Maturation, and Disease

Publisher Summary This chapter presents an overview of myosin isoenzyme transitions in muscle development, maturation, and disease. Myosin is the major contractile protein found in all muscle cells and is present in most nonmuscle cells as well. Like other ubiquitous proteins, myosin is represented by a multigene family. The diversity of myosins from muscle and nonmuscle cells is quite large. This chapter describes the characterization and regulation of myosin in skeletal, cardiac, and smooth muscles with particular emphasis on the transitions of myosin isoforms that occur during normal development and maturation. Many diseases of muscle can affect myosin composition or interfere with the transitions of myosin isoforms. The molecular structure of most myosins is quite similar, consisting of two globular heads attached to a-helical rodlike tail. The subunit structure of myosin consists of two heavy chains (MHC) of molecular weight 200,000 and 4 mol of light chains of molecular weight 18,000–26,000.

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.

An immunological method to assess protein degradation in post-mortem muscle.

A method for determining proteolysis of any specific protein in muscle is demonstrated. The protocol involves the preparation of a specific antibody which is used in immunoblotting total protein extracts from post-mortem muscle. In the present study an antibody to bovine myosin heavy chain was prepared that reacts with intact myosin heavy chain as well as proteolytic fragments that are generated by proteases. We show that little, if any, myosin heavy chain fragments are generated during prolonged aging of muscle at 4°C. In contrast, storage of muscle at 37°C results in the rapid breakdwon of myosin heavy chain to immunologically detectable peptides. Using a monoclonal antibody to titin, we demonstrate that this protein is degraded at 4°C during the aging period, and that, between 2 and 3 weeks following slaughter, no undergraded titin is detectable. This method is suitable for the analysis of any protein that can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens☆

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.

Analysis of the chicken fast myosin heavy chain family: Localization of isoform-specific antibody epitopes and regions of divergence☆

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.

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 dot‐blot 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. Dev. Dyn. 208:491–504, 1997.

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. Microsc. Res. Tech. 50:473–491, 2000.

Myoblasts from fetal and adult skeletal muscle regulate myosin expression differently

We compared the expression of myosin heavy chains in myogenic cultures prepared from fetal (embryonic Day 10) and adult (12-16 weeks) chicken pectoralis muscle using immunofluorescence with isoform-specific monoclonal antibodies. We found that the majority of fetal myocytes (differentiated myoblasts) and myotubes coexpressed ventricular and embryonic myosin heavy chains in culture. Also, when fetal cells were plated at a clonal density most clones coexpressed both ventricular and embryonic isoforms. In contrast, all adult myocytes and newly formed adult myotubes expressed just ventricular myosin, whether plated at mass or clonal densities. Within 12-24 hr of the onset of fusion, adult myotubes began to express embryonic myosin as well. Eventually, the majority of adult myotubes coexpressed both ventricular and embryonic myosin. The delay of embryonic myosin expression until after fusion was also seen in passaged adult myoblasts and in myoblasts isolated from regenerating adult muscle. The expression of embryonic myosin can be abolished by inhibiting fusion with EGTA in adult but not in fetal cultures. We conclude that both fetal and adult myotubes express ventricular and embryonic myosins but only fetal myocytes express the embryonic isoform prior to fusion. This difference in the regulation of embryonic myosin expression between fetal and adult myoblasts supports the hypothesis that these cells may represent two distinct populations of myogenic precursors.