Isolation and characterization of two molecular variants of myosin heavy chain from rabbit ventricle. Change in their content during normal growth and after treatment with thyroid hormone

We have prepared monoclonal antibodies specific for cardiac myosin heavy chain. These antibodies were used for the separation and characterization of the molecular variants of myosin heavy chain present in the rabbit heart. Two molecular forms of myosin heavy chain, HC alpha and HC beta, were isolated from the euthyroid rabbit heart by affinity chromatography. Their reactivity with our antibodies indicated that the primary structures of HC alpha and HC beta differ in at least four and share at least two antigenic determinants. Differences in the primary structure of HC alpha and HC beta were confirmed by analysis of the peptides produced by limited chymotryptic digestion of the two heavy chains. Thirteen peptide differences were consistently found. The HC alpha and HC beta variants are shown by immunologic analysis and in chymotryptic peptide profiles to be identical with the predominant forms of myosin heavy chain synthesized in the hearts of hyperthyroid and adult euthyroid rabbits, respectively. During development and maturation of the euthyroid rabbit heart, HC alpha comprises approximately 50% of the ventricular myosin between birth and 4 weeks of age; it diminishes to 20-30% by 8 weeks and to 10-20% by 12 weeks of age. Cardiac myosin from a 1-year-old rabbit is composed almost entirely of HC beta. Cardiac myosin from embryonic animals at 20 days gestation contained 20% HC alpha. These results show that HC alpha occurs normally in the euthyroid rabbit heart and that the relative proportions of HC alpha and HC beta depend on both the developmental stage and the thyroid state of the animal.     

Enzymatic comparisons between light chain isozymes of human cardiac myosin subfragment-1

Human cardiac ventricular myosin subfragment-1 (S-1) was prepared by chymotryptic digestion of myosin purified from adult and fetal hearts. The enzymatic properties of adult S-1 were compared to those of two light chain isozymes of fetal S-1 which were separated by ion-exchange chromatography. One fetal isozyme contained a light chain (LC) indistinguishable from the adult ventricular LC1 and the other fetal isozyme contained the LC1 variant that is a component of intact fetal myosin. The fetal isozymes had identical actin-activated Mg2+ ATPase rates at all actin concentrations, as well as the same K+EDTA, Ca2+, and Mg2+ATPase rates. Furthermore, both fetal isozymes had the same actin-activated Mg2+ATPase rates as S-1 purified from adult hearts. The K+EDTA and Ca2+ATPase rates of adult S-1 were only slightly different from those of fetal S-1. These observations are consistent with other available data suggesting that human fetal and adult ventricular myosin differ only in light chain content, not in heavy chain composition, and indicate that isozymic LC1 variation does not alter the steady-state ATPase rate of human cardiac S-1.     

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.     

Comparison of myosin heavy chains in atria and ventricles from hyperthyroid, hypothyroid, and euthyroid rabbits

The structural similarities of the heavy chains (HC) of myosin isolated from atria and ventricles of hyper-, hypo-, and euthyroid rabbits were compared by immunological analysis, by one- and two-dimensional peptide mapping, and by electrophoresis under nondenaturing conditions. Monoclonal and polyclonal antibodies, which are specific for HC alpha of ventricular myosin, cross-reacted equally with the HCs of euthyroid atrial myosin. Our immunological analysis identified multiple epitopes common to euthyroid atrial HC and ventricular HC alpha. One- and two-dimensional gel electrophoretic analysis of peptides produced by partial proteolytic digestion of each type of HC reveal no differences between euthyroid atrial HCs and ventricular HC alpha, whereas marked differences are apparent between atrial HC and ventricular HC beta. Nondenaturing gel electrophoresis separates ventricular myosin from hyper- and hypothyroid rabbits into two forms, V1 and V3, respectively. In euthyroid atria, two isomyosins, A1 and A2, were resolved; with the slower moving A2 isomyosin having the same mobility as that of the V1 isomyosin. After cross-hybridization of light chains of ventricular myosin with euthyroid atrial HCs, only a single band having a mobility identical with that of the V1 isomyosin was seen. Furthermore, atrial myosin HCs isolated from hyper- and hypothyroid rabbits were indistinguishable from euthyroid atrial HC and from ventricular HC alpha by these procedures. We conclude that ventricular HC alpha and atrial HC are indistinguishable proteins, and that the type of HC expressed in the atria is unaffected by the thyroid state of the rabbit.     

Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1

Summary Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V₁ isomyosin) has been prepared and purified by DEAF-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca²⁺, Mg²⁺ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1.The differences between these subfragments are due to the differences in the light chains associated with them.Abbreviations EDTA Ethylene Diamine Tetra-acetic Acid - SDS Sodium Dodecyl Sulfate - S₁ myosin subfragment-1 - HC Heavy Chain - LC Light Chain     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Myosin heavy chain in avian muscular dystrophy corresponds to the neonatal isozyme

We have previously demonstrated, based on comparison of homologous amino acid sequences and of two-dimensional CNBr peptide gel patterns, that the myosin heavy chain in pectoralis muscles of Storrs, Connecticut dystrophic chickens is different from that of their normal controls (Huszar, G., Vigue, L., De-Lucia, J. Elzinga, M., and Haines, J. (1985) J. Biol. Chem. 260, 7429-7434). Others have shown, however, that genomic banks and mRNA complements of the control and dystrophic birds are not different. In the present studies, we have examined the hypothesis that the "dystrophic" myosin heavy chain is not a novel gene product, but is a developmental isozyme which is expressed in pectoralis muscles of adult chickens due to the dystrophic process. Two-dimensional maps of myosin heavy chain CNBr peptides were prepared from breast muscles of 17-day in ovo (embryonic), 25-day posthatch (neonatal), and adult birds of the Storrs dystrophic and of two control strains. Also, myosin and actomyosin ATPase enzymatic activities of the various preparations were determined in the pH range of 5.5 to 9.0. Analysis of the peptide maps demonstrates that the embyronic, neonatal, and control adult myosin heavy chain isozymes are distinctly different gene products with only minute variations between the respective developmental isozymes in dystrophic and control muscles. However, the pectoralis myosin heavy chain of adult dystrophic birds, which is a homogeneous isozyme population by amino acid sequences and gel patterns, corresponds to that of the neonatal-type myosin heavy chain. The ATPase properties of the embryonic, neonatal, or adult pectoralis myosins and actomyosins were not different, whether the level of specific activity or the pattern of pH activation is considered. Since the mobility of neonatal chicks (primarily neonatal-type isozymes) is not restricted, the differences in myosin heavy chain structures are part of the syndrome, but not the cause of avian muscular dystrophy.     

Immunological and biochemical evidence for atrial-like isomyosin in thyrotoxic rabbit ventricle

Immunological, structural and enzymatic characteristics of atrial and ventricular myosin from euthyroid rabbits were analyzed and compared with ventricular myosin from hyperthyroid animals. (1) Specific antibodies against bovine atrial myosin were found to react selectively in double-immunodiffusion and enzyme immunoassay with both rabbit atrial myosin and ventricular myosin from thyroxine-treated animals. These specific anti-bovine atrial myosin antibodies reacted with the heavy chains of thyrotoxic ventricular myosin when examined by enzyme immunoassay combined with SDS-polyacrylamide gel electrophoresis. (2) In one-dimensional SDS-polyacrylamide gel electrophoresis, no difference could be demonstrated in the light chain pattern of ventricular myosin from euthyroid and hyperthyroid rabbit hearts. One-dimensional analysis of myosin heavy chains after chymotryptic digestion in the presence of SDS showed significant differences between the two ventricular myosins. Also, the peptide maps from atrial myosin resembled the pattern of peptides found with ventricular heavy chains from hyperthyroid rabbits. The steady state rate, the alkali stability and the pH sensitivity of Ca2+-ATPase activity of thyrotoxic ventricular myosin were very similar to those of atrial myosin. (3) These results provide direct immunochemical and biochemical evidence for the existence of an atrial-like isomyosin in thyrotoxic rabbit ventricles.     

Analysis of cloned mRNA sequences encoding subfragment 2 and part of subfragment 1 of alpha- and beta-myosin heavy chains of rabbit heart

Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.     

Light chains of chicken embryonic gizzard myosin.

1. Myosin from gizzards of 15-day-old chicken embryos was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultra-centrifugation and Sepharose 4B chromatography. 2. The myosin composed of heavy and three light chains as determined by sodium dodecyl sulfate (SDS) gel electrophoresis. The molecular weights of the light chains were 23,000 (L23), 20,000 (L20), and 17,000 (L17), respectively. The amount of L23 light chain decreased and disappeared, and the L17 light chain increased steadily in the course of development. The amount of L20 light chain did not change. 3. ATPase activity of the embryonic myosin was essentially the same as that of adult myosin. The change in the light chain pattern in the course of development did not correlate to the ATPase activity. 4. Antigenicity of the heavy chains in the embryonic myosin was the same as that of the adult heavy chains. However, antibodies to light chains were not detected in the antibodies to either the embryonic or adult myosins.     

Relation of fast and slow skeletal, and atrial and ventricular myosin in various mammals

Myosin was isolated from adult mouse, rat, rabbit and cat atrial and ventricular myocardium and fast and slow skeletal muscles and examined by measuring Ca2+-ATPase activity and by electrophoretic fractionation of chymotryptic peptides and MLCs. The myosin from mouse atrial and ventricular myocardium were very similar. The properties of cat soleus muscle myosin and ventricular myocardium were also very similar (ATPase activity and electrophoretic pattern of chymotryptic peptides of myosin). The electrophoretic pattern of MLCs, however, was distinct when comparing mouse and feline muscles. These observations are consistent with the idea that atrial and ventricular alpha MHCs are closely related and that beta MHCs from ventricular myocardium and slow skeletal muscle fibres are also closely related.     

Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.     

Chronic oral administration of beta-adrenoceptor agonist clenbuterol affects myosin heavy chain (MHC) expression in adult mouse heart

The aim of this study was to analyze the effects of chronic administration of the beta-adrenoceptor agonist clenbuterol (2 mg/kg body weight/day for a period of 30 days) on the major contractile protein (myosin) in the left ventricular muscle of the adult mouse heart. Separation of myosin heavy chain (MHC) isoforms on 7.5 % glycerol SDS-PAGE and subsequent quantification of the gels by laser densitometry showed a 6.5-fold increase in the beta-isoform of MHC in the clenbuterol-treated group. The alpha : beta ratio of these two isoforms in the control group was 98.16+/-0.14 %: 1.83+/-0.14 %, whereas in the treated group it was 88.05+/-1.15 % : 11.95+/-1.15 %. Actomyosin ATPase activity assay demonstrated a significant (20 %) decline in ATPase activity of the tissue in the beta-agonist-treated group. These results suggest that chronic clenbuterol treatment is capable to induced the transformation of MHC isoforms increasing the slow beta-MHC isoform, which may contribute to the altered contractile mechanics of clenbuterol-treated hearts.     

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.     

Distribution and structure-function relationship of myosin heavy chain isoforms in the adult mouse heart

The two cardiac myosin heavy chain isoforms, alpha and beta, exhibit distinct functional characteristics and therefore may be distributed regionally within the heart to match the functional demands of a specific region. In adult mouse hearts, which predominantly express alpha-myosin heavy chain, we observed high concentrations of beta-myosin in distinct areas such as at the tip of papillary muscles and at the base close to the valvular annulus. In light of these distinct distribution patterns of the myosin isoforms, we subsequently explored the isoform-specific structure-function relationships of the myosins. The alpha- and beta-isoforms are 93% identical in amino acid sequence, but it remains unclear which of the nonidentical residues determines isoform functionality. We hypothesized that residues situated within or close to the actin-binding interface of the myosin head influence actin binding and thereby modulate actin-activated ATPase activity. A chimeric myosin was created containing beta-sequence from amino acid 417 to 682 within the alpha-backbone. In mice, approximately 70% of the endogenous cardiac protein was replaced with the chimeric myosin. Myofibrils containing chimeric myosin exhibited ATPase activities that were depressed to the levels observed in hearts expressing approximately 70% beta-myosin. In vitro motility assays showed that the actin filament sliding velocity generated by chimeric myosin was similar to that of alpha-myosin, almost twice the velocities observed with beta-myosin. These data indicate that this large domain sequence switch conferred beta-like actin-activated ATPase activities to the chimeric myosin, suggesting that this region is responsible for the distinct hydrolytic properties of these myosin isoforms.     

Correlation of myofibrillar ATPase activity and myosin heavy chain content in ventricular and atrial myocardium of fish heart

In the fish heart, ventricular and atrial muscles contain different isoforms of native myosin and myosin heavy chain (MyHC) but the significance of this diversity is still not known. We have analysed ventricular and atrial myocardium of six freshwater fish species (goldfish, roach, bream, rudd, perch and pike-perch) using histochemical staining for myofibrillar ATPase activity as well as non-denaturing and SDS gel electrophoreses for native myosin and MyHC content. In the range of fish species studied, the intensity of ATPase reaction was higher in the atrial myocardium than in the ventricular myocardium and the composition of native myosin isoforms differed between these two muscles. The MyHC content in the cardiac muscle showed some species-related differences. In the goldfish, both atrial and ventricular cardiac muscle contained electrophoretically similar MyHC. In the other fish species, however, the ventricular myocardium showed electrophoretically faster MyHC than that present in the atrial myocardium. These results indicate that there are consistent and characteristic species-related differences between the ventricular and atrial muscles at the level of ATPase staining and the type of MyHC expressed. The findings suggest that fish ventricular and atrial muscles may differ in their contractile properties.     

Expression of myosin heavy chains during thyroid hormone-induced cardiac growth

The expression of mRNAs for two cardiac myosins has been studied in the ventricles of hypo- and hyperthyroid rabbits by using cloned cDNA sequences corresponding to the mRNAs of the alpha- and beta-myosin heavy chains (HCs). The temporal change in relative levels of the alpha and beta HC mRNAs after triiodothyronine (T3) treatment of hypothyroid rabbits was determined by nuclease S1 mapping. In the hypothyroid state, only NC beta-mRNA was expressed in the ventricles. The HC alpha-mRNA was first detectable 4 h after administration of T3 (200 micrograms/kg) to hypothyroid animals. By 12, 24, and 72 h, HC alpha-mRNA represented 20, 50, and 90% of total myosin mRNA. The relationship between the relative mRNA levels and relative synthesis rates of myosin HCs was evaluated in 5- to 6-wk-old normal and thyrotoxic rabbits. Myosin synthesis rates were determined by labeling of protein in vivo with [2H]leucine. The V1 (HC alpha) and V3 (HC beta) isomyosins were separated by immune affinity chromatography and the HCs were isolated electrophoretically. In a normal euthyroid group of animals and in animals 12 and 24 h after administration of 200 micrograms of thyroxine, the relative mRNA levels and relative synthesis rates of the alpha and beta HCs were not significantly different. Our results show that, first, thyroid hormone causes a rapid accumulation of HC alpha-mRNA and loss of HC alpha-mRNA, and second, in normal and thyrotoxic rabbits, the relative synthesis rates of HC alpha and HC beta reflect the relative abundance of their respective mRNAs. These data are consistent with the thyroid hormones regulating synthesis of ventricular myosin at steps that precede translation of its message.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.