Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.     

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

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.     

Comparison of myosin isoenzymes present in skeletal and cardiac muscles of the Arctic charr Salvelinus alpinus (L.). Sequential expression of different myosin heavy chains during development of the fast white skeletal muscle.

The expression of myosin isoforms and their subunit composition in the white skeletal body musculature of Arctic charr (Salvelinus alpinus) of different ages (from 77-day embryos until about 5 years old) was studied at the protein level by means of electrophoretic techniques. Myosin from the white muscle displayed three types of light chain during all the developmental stages examined: two myosin light chains type 1 (LC1F) differing in both apparent molecular mass and pI, one myosin light chain type 2 (LC2F) and one myosin light chain type 3 (LC3F). The fastest-migrating form of LC1F seemed to be predominant during the embryonic and eleutheroembryonic periods. The slowest-migrating form of LC1F was predominant in the 5-year-old fish. Between 1 year and 4 years, both types of LC1F were present in similar amounts. Cardiac as well as red muscle myosin from 3-year-old fish had two types of light chain. The myosin light chains from atria and ventriculi were indistinguishable by two-dimensional electrophoresis, but were different from the myosin light chains from red muscle. Neither the light chains from cardiac nor red muscle were coexpressed with the myosin light chains of white muscle at any of the developmental stages examined. Two myosin heavy chain bands were resolved by SDS/glycerol/polyacrylamide gel electrophoresis of the extract from embryos. One of the bands was present in minor amounts. The other, and most abundant, band comigrated with the only band found in the extracts of white muscle myosin from older fish. One-dimensional Staphylococcus aureus V8 protease peptide mapping of these bands revealed some differences during development of the white muscle tentatively interpreted as follows. The myosin heavy chain band present in minor amounts in the embryos may represent an early embryonic form that is replaced by a late embryonic or foetal form in the eleutheroembryos. The foetal myosin heavy chain appears to be present until the resorption of the yolk sack and beginning of the free-swimming stage. A new form of myosin heavy chain, termed neonatal and probably expressed around hatching, is present until about 1 year of age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.     

Myosin heavy chain isoforms in white, red and ventricle muscles of barbel (Barbus barbus L.).

1. Actomyosin extracts of trunk, heart, and head muscles from barbel (Barbus barbus L.) were analyzed by SDS-polyacrylamide gel electrophoresis to study their myosin heavy chain composition. 2. Four heavy chain isoforms were found: trunk white, trunk red, and ventricle muscles yielded one heavy chain typical of the muscle type; head muscles devoid of red fibers displayed two heavy chain isoforms, the slow migrating one corresponding to the trunk white muscle type. 3. The electrophoretic mobility of red and ventricle myosin heavy chains related to that of white isoforms appeared highly modified by the glycerol content of the gels.     

Energy metabolism of carp swimming muscles

Summary Electromyography has been used to study the recruitment of red, pink and white muscle fibres of the Mirror carp at different swimming speeds. Locomotion below 0.3–0.5 L/S (lengths per second) is achieved primarily by fin movements after which the red myotomal muscle becomes active. Pink muscle fibres are the next type to be recruited at speeds around 1.1–1.5 L/S. White muscle is only used for fast cruising in excess of 2–2.5 L/S and during bursts of acceleration.Studies of the myofibrillar ATPase activities of these muscles have shown a ratio of 124 for the red, pink and white fibres respectively. The myosin low molecular weight components, which are characteristic of the myosin phenotype, have been investigated by SDS polyacrylamide electrophoresis. The light chain patterns of the pink and white muscles were identical and characteristic of the fast myosin phenotype. Red muscle myosin had a light chain pattern characteristic of slow muscles. It would appear that there is a relationship between the speed of contraction of the fibre types and the locomotory speed at which they are recruited.The activities of some enzymes of energy metabolism have also been determined in the three muscle types. Enzymes associated with oxidate metabolism have high, intermediate and low activities in the red, pink and white muscles respectively. Pyruvate kinase and lactate dehydrogenase activities were considerably higher in the pink than in either red or white muscles. It is suggested that the high capacity for anaerobic glycolysis of the pink muscle is associated with its recruitment for sustained effort at swimming speeds above which the fish can no longer meet all its energy requirements by gas exchange at the gills.Abbreviations used EDTA ethylenediamine tetraacetic acid - L/S lengths, sec^–1 - LDH Lactate dehydrogenase - PFK phosphofructokinase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Purification of messenger ribonucleic acids for fast and slow myosin heavy chains by indirect immunoprecipitation of polysomes from embryonic chick skeletal muscle

Fast and slow myosin heavy chain mRNAs were isolated by indirect immunoprecipitation of polysomes from 14-day-old embryonic chick leg muscle. The antibodies were prepared against myosin heavy chains purified by NaDod-SO4-polyacrylamide gel electrophoresis and were shown to be specific for fast and slow myosin heavy chains. The RNA fractions directed the synthesis of myosin heavy chains in a cell-free translation system from wheat germ. Several smaller peptides were also synthesized in lower concentrations. These probably are partial products of myosin heavy chains, since they are immunoprecipitated with antibodies to myosin heavy chains. Immunoprecipitation of the translation products with the antibodies to fast and slow myosin heavy chains showed the RNA preparations to be approximately 94% enriched for fast myosin heavy chain mRNA and approximately 84% enriched for slow myosin heavy chain mRNA with respect to myosin HC type. Peptides having slightly different mobilities on NaDodSO4-polyacrylamide gels were immunoprecipitated by antibodies to fast and slow myosin heavy chains.     

White muscle differentiation in the eel (Anguilla anguilla L.): changes in the myosin isoforms pattern and ATPase profile during post-metamorphic development

Myosin isoforms and their light and heavy chains subunits were studied in the white lateral muscle of the eel during the post metamorphic development, in relation with the myosin ATPase profile. At elver stage VI A1 the myosin isoforms pattern was characterized by at least two isoforms, FM3 and FM2. The fast isomyosin type 1 (FM1) appeared during subsequent development. It increased progressively in correlation with the increase in the level of the light chain LC3f. FM1 became predominant at stage VI A4. At the elver stage VI A1, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed at least two heavy chains, namely type II-1 and II-2. The type II-1 heavy chain disappeared in the yellow eel white muscle, and V8-protease peptide map showed the appearance of a minor heavy chain type II-3 as early as stage VI B. Comparison of myosin heavy chains and myosin isoforms patterns showed the comigration of different myosin isoforms during white muscle development. The myosin ATPase profile was characterized by a uniform pattern as far as stage VI A4. A mosaic aspect in white muscle was observed as early as stage VI B, showing the appearance of small acid labile fibers. This observation suggests that the type II-3 heavy chain is specific to the small fibers.     

Histochemical fibre types in the lateral muscle of fishes in fresh, brackish and salt water

The histochemical pattern of red, pink and white muscle of fish living in fresh, brackish, and salt water is reported. The muscle fibres were stained routinely during the year for lactate dehydrogenase (LDH), menadione α-glycerophosphate dehydrogenase (Mα—GPDH), succinic dehydrogenase (SDH), myosin adenosine triphosphatase (myosin ATPase), phosphorylase, lipids and glycogen. The pink and red muscles contain more glycogen and lipids and have a higher SDH activity, which is in accord with their aerobic metabolism and function in sustained swimming activity. The acid labile myosin ATPase activity characteristic of fast twitch fibres is present in the white fibres of most species, however in the white muscle of Gobius paganellus the enzyme activity is stable to both acid and alkali and, in addition, there is a scattered distribution of different fibre types in red and, especially, pink muscle. A study of seasonal variation patterns of myosin ATPase in white muscle of mugilidae over a period of two years has demonstrated, in late summer, the appearance of new small diameter fibres, with a high acid stable enzyme activity, that develop into the large diameter acid labile fibres.     

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.     

The analysis of a chicken myosin heavy chain cDNA clone

A cDNA library has been constructed in the plasmid pBR322 using a large size class of RNA derived from chicken embryonic leg muscle as the template material. A clone containing a 2350-base pair insert was selected and identified as coding for the myosin heavy chain sequence, based upon its ability to hybridize to genomic myosin heavy chain clones, and by direct nucleotide sequencing. Cross-hybridization experiments with myosin heavy chain genomic clones, and mRNAs derived from different muscle types were used to explore the heterogeneity of the various myosin heavy chain isoforms at the level of the coding sequences. Although extensive sequence homology with the other isoforms was observed, a fast white isoform-specific subclone was constructed, and used to demonstrate that different genes code for the adult and embryonic fast white myosin heavy chain proteins.     

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.     

Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.