Synthesis of adult myosin light chains by embryonic muscle cultures.

Myosin light chain synthesis has been analyzed in cultures of fast and slow muscles from chicken and quail embryos. Synthesis was assayed by [35S]methionine incorporation and two-dimensional electrophoresis of total cell extracts. Our results show that differentiated cultures of embryonic anterior latissimus dorsi and pectoral muscles synthesize proteins that comigrate on two-dimensional gels with the five myosin light chains of adult fast (pectoral) and slow (anterior latissimus dorsi) muscle. Partial proteolytic digestion and peptide analyses further confirm the identity of these proteins as adult light chains. Cultures of dividing myoblasts do not synthesize any of these fiber type isozymes, and synthesis of the isozymes is initiated at myoblast fusion. Also, myogenic clones drived from single myoblasts differentiate to synthesize these five myosin light chains, indicating that individual myoblasts have the potential to express the synthesis of all fiber type light chain isozymes. We conclude that the primary events in muscle differentiation include the initiation of synthesis of the entire set of adult fast and slow myosin light chain isozymes. The developmental and physiological implications of these results for the establishment of fiber type specificity are discussed.     

Myosin types during the development of embryonic chicken fast and slow muscles.

We have studied the myosin types present in developing fast and slow muscles of the chicken embryo. Myosin light chains were characterized by their mobility on sodium dodecyl sulfate/polyacrylamide gels; myosin heavy chains were identified by their reaction with antibodies specific for adult fast or adult slow myosin heavy chains. During development, the pectoralis muscle, a fast muscle in the adult, contains heavy chains and two of the three light chains characteristic of adult fast muscle myosin. However, the anterior latissimus dorsi muscle, a slow muscle in the adult, also contains fast myosin light and heavy chains during early development. Only after the time of innervation does this muscle begin synthesizing predominantly the slow myosin heavy and light chains. We hypothesize that the synthesis of fast myosin in both early fast and slow muscles is the result of the endogenous program for muscle development; initiation of the synthesis of slow myosin, however, is dependent upon exogenous factors.     

Immunofluorescence analysis of the primordial myosin detectable in embryonic striated muscle.

Immunofluorescence analysis showed that the earliest myosin detectable in both the embryonic chicken heart and somitic myotome, the precursor to skeletal muscle, was strongly reactive with two different monoclonal antibodies specific for the heavy chain of cardiac ventricular myosin, but it showed no reactivity with affinity-purified polyclonal antibodies specific for the heavy chains of either fast-twitch or slow-tonic skeletal myosins. The heart remained reactive exclusively with the antibodies to cardiac myosin throughout development, while late embryonic (day 20) skeletal muscles were strongly reactive only with their homologous skeletal myosin antibodies. Our findings suggest that the primordial myosin heavy chain detectable in both forms of embryonic chicken striated muscle, the myotome and the heart, is immunologically distinct from myosins expressed in later embryonic as well as adult skeletal muscles, but it contains antigenic determinants similar to those present in cardiac ventricular myosin.     

Atrial and ventricular myosins from human hearts. I. Isoenzyme distribution during development and in the adult

Summary Two distinct types of native isomyosins, referred to as human fetal HF and human ventricular myosin HV-3, have been identified in the human fetal heart during two different periods of gestation and in the neonatal state, whose relative parts change with development. In the adult ventricle, only HV-3 was found. Two myosin isoforms designated as HA-3 and HA-1 occur in the atrial myocardium of the normal human heart, which are electrophoretically distinct from the fetal isoenzymes.In fetal tissue, the myosin light chain complement is composed of atrial and ventricular light chains. In support of recent results, we also found identical spots from the atrial ALC-1 and the fetal light chain FLC, suggesting a homology between them. Apart from the light chains typical for this tissue, the atrial myocardium also contains ventricular light chains. Therefore we hypothesize that atrial myosin consists of two atrial isoenzymes and presumably of a ventricular type, too.Differences between atrial and ventricular myosin from human hearts were demonstrated by measuring the temperature dependence of the Ca²⁺-ATPase.     

Myosin light chains and the developmental origin of fast muscle.

Physiological characteristics of embryonic and fetal fast muscle function are similar to those of adult slow muscles, whereas most biochemical data suggest that embryonic and fetal fast muscles contain only fast muscle myosin. In the studies reported here, myofibrillar preparations from developing avian pectoral muscle (fast muscle) were isolated and analyzed for myosin light-chain type and synthesis. These analyses show that early in development avian fast muscle synthesizes and assembles myofibrils with light chains of both slow and fast myosins. Later in development, fast muscle no longer assembles myofibrils containing slow myosin light chains due to the cessation of synthesis of slow myosin light chains in mid-development. These in vivo studies indicate that the more developmentally primitive type of skeletal muscle is one that synthesizes both slow and fast myosin light chains independent of its anatomic location, and an event(s) late in fast muscle development results in the repression of synthesis of slow myosin light chains.     

Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments.

Dictyostelium myosin is composed of two heavy chains and two pairs of light chains in a 1:1:1 stoichiometry. Myosin purified from amoebae grown in medium containing [32P]phosphate had two of the subunits labeled (0.2-0.3 mol of phosphate per mol of 210,000-dalton heavy chains and approximately 0.1 mol of phosphate per mol of 18,000-dalton light chain). Kinase activities specific for the 210,000-dalton and for the 18,000-dalton subunits have been identified in extracts of Dictyostelium amoebae, and the heavy chain kinase has been purified 50-fold. This kinase phosphorylated Dictyostelium myosin to a maximum of 0.5-1.0 mol of phosphate per mol of heavy chain. Heavy chain phosphate, but not light chain phosphate, can be removed with bacterial alkaline phosphatase. Actin-activated myosin ATPase increased 80% when phosphorylated myosin was dephosphorylated to a level of approximately 0.06 mol of phosphate per mol of heavy chain. This effect could be reversed by rephosphorylating the myosin. The ability of myosin to self-assemble into thick filaments was inhibited by heavy chain phosphorylation. For example, in 80-100 mM KCl, only 10-20% of the myosin was assembled into thick filaments when the heavy chains were fully phosphorylated. Removal of the heavy chain phosphate resulted in 70-90% thick filament formation. This effect on self-assembly could be reversed by rephosphorylating the dephosphorylated myosin. These findings suggest that heavy chain phosphorylation may regulate cell contractile events by altering the state of myosin assembly.     

Comparison of adult, embryonic, and dystrophic myosin heavy chains from chicken muscle by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and peptide mapping.

Chicken myosin heavy chains from adult fast white muscle fibers (both normal and dystrophic), adult slow red fibers, and embryonic presumptive fast white fibers were compared by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by peptide mapping. The heavy chain of slow red myosin migrated electrophoretically more slowly than the heavy chains of the other myosins and differed markedly from them in its peptide maps. The heavy chain of dystrophic fast white myosin was similar to its normal counterpart by peptide mapping but showed slight differences. The peptide map of the heavy chain of embryonic presumptive fast white myosin had the general features of that of the heavy chain of fast white, not slow red, fibers but contained definite differences from the former. The results are consistent with the existence of a separate gene for the heavy chain of embryonic presumptive fast white myosin.     

Biochemical characteristics of human cardiac myosin.

Myosin was purified from the left ventricles of eight patients. Samples of the ventricle were obtained immediately after the death of three patients whereas the rest were obtained from $\text{2}\text{1}\text{2}$ to 20 h after the death. Human cardiac myosin, like the cardiac myosin from other mammalian species has only two light chains. The corresponding molecular weights were 26000 and 20000 for light chain 1 (LC₁) and light chain (LC₂) respectively. In its chemical properties, total sulfhydryl groups and responses to solvents the human cardiac myosin resembles canine cardiac myosin. These studies also show that a delay in the preparations of myosin up to 20 h does not alter its properties. ATPase activities (Ca²⁺, Mg²⁺ and K⁺-EDTA) did not show any correlation with the age of the subject. However the total-SH content was significantly lower in the elderly patients.     

cDNA recombinant plasmid complementary to mRNAs for light chains 1 and 3 of mouse skeletal muscle myosin.

A recombinant plasmid with a cDNA sequence transcribed from mouse skeletal muscle RNA is shown to hybridize with mRNAs for myosin light chains LC1F and LC3F. The inserted fragment corresponds exclusively to the 3'-noncoding region of the mRNA. It hybridizes almost exclusively with the two light chain messengers from fast skeletal muscle RNA of adult mouse. Slight hybridization is seen with RNA from heart muscle and embryonic skeletal muscle. The implications of the conservation of the 3'-noncoding regions between the two mRNAs are discussed.     

Comparative sequence of myosin light chains from normal and hypertrophied human hearts

Myosin light chains from normal and hypertrophied human hearts were partially sequenced in order to see whether structural modifications of these light subunits could provide a molecular basis for the changes observed in heart properties and in myosin enzymatic activity. Normal light chains were prepared form hearts taken at autopsy, weighing 350 g or less and apparently devoid of myocardial disease. "Hypertrophied cardiac myosin light chains" were prepared from two greatly hypertrophied hearts, weighing 600 and750 g. No amino acid substitutions, deletions, or additions were observed in the light chains from hypertrophied hearts. The third light chain previously reported in human cardiac myosin and related to hypertrophy was found to be a proteolytic product of LC2. The comparison between human and beef cardiac myosin light chains indicated that the sequences of these subunits of the myosin molecule are highly conserved.     

Contractile protein isozymes in muscle development: identification of an embryonic form of myosin heavy chain.

The nature of the myosin heavy chain in embryonic muscle tissue, cultured muscle cells, and several adult muscles was investigated. After denaturation with sodium dodecyl sulfate, purified rat myosins were subjected to partial proteolytic cleavage or immunological analysis using microcomplement fixation. Three types of myosin heavy chains could be demonstrated by both approaches. Whereas adult muscles contain fast- or slow-type myosin heavy chains, embryonic tissue and cultured muscle cells harbor a distinct embryonic form. The existence of this distinct form further characterizes the isozymic transitions of contractile proteins during muscle development.     

Coevolution of head, neck, and tail domains of myosin heavy chains

Myosins, a large family of actin-based motors, have one or two heavy chains with one or more light chains associated with each heavy chain. The heavy chains have a (generally) N-terminal head domain with an ATPase and actin-binding site, followed by a neck domain to which the light chains bind, and a C-terminal tail domain through which the heavy chains self-associate and/or bind the myosin to its cargo. Approximately 140 members of the myosin superfamily have been grouped into 17 classes based on the sequences of their head domains. I now show that a phylogenetic tree based on the sequences of the combined neck and tail domains groups 144 myosins, with a few exceptions, into the same 17 classes. For the nine myosin classes that have multiple members, phylogenetic trees based on the head domain or the combined neck/tail domains are either identical or very similar. For class II myosins, very similar phylogenetic trees are obtained for the head, neck, and tail domains of 47 heavy chains and for 29 essential light chains and 19 regulatory light chains. These data strongly suggest that the head, neck, and tail domains of all myosin heavy chains, and light chains at least of class II myosins, have coevolved and are likely to be functionally interdependent, consistent with biochemical evidence showing that regulated actin-dependent MgATPase activity of Dictyostelium myosin II requires isoform specific interactions between the heavy chain head and tail and light chains.     

Myosin isozymes in rabbit and human smooth muscles

Although multiple forms of myosin in cardiac and skeletal muscles have been identified, it has not been firmly established that myosin isozymes are present in adult smooth muscle. Myosin, extracted from human thoracic aorta and lower saphenous vein and rabbit aorta and uterus, was analyzed by pyrophosphate gel electrophoresis to determine if myosin isozymes are present in these tissues. In all smooth muscle tissues studied, two myosin isozymes were detected and labelled as smooth muscle 1 and smooth muscle 2, smooth muscle 2 being the faster migrating isozyme. Bovine cultured smooth muscle cells from the media of thoracic aorta also contained two forms of myosin. However, cultured fibroblasts contained only one form of myosin. Extracting myosin from either relaxed or contracting rabbit aortic smooth muscle did not influence the mobilities of smooth muscle 1 and smooth muscle 2 on pyrophosphate gels, suggesting that the degree of light chain phosphorylation did not significantly alter the electrophoretic mobility under our conditions. Smooth muscle 1 and smooth muscle 2 myosins each contain heavy chains (200,000 daltons) and light chains (20,000 and 17,000 daltons) in addition to filamin (235,000 daltons), which is closely associated with the native protein. Myosin peptide maps of rabbit aorta and uterus revealed areas of substantially different banding patterns between smooth muscle 1 and smooth muscle 2 from the same tissue. Similar peptide maps of smooth muscle 1 bands were produced from the different tissues, but the smooth muscle 2 maps were dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of ventricular myosin subunits in the atria of children with congenital heart malformations

The presence of ventricular myosin light chains in the atria of children with congenital heart disease was demonstrated by two-dimensional polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis. Ventricular myosin light chains were present in 27% of biopsies from 91 children with different forms of congenital heart disease. Perimembranous ventricular septal defects and tetralogy of Fallot were associated with the presence of ventricular myosin light chains in 50% of patients. The presence of ventricular myosin light chains in these atria did not correlate with pressure or volume overload. Analysis of myosin heavy chain isotype in the same biopsies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis indicated that there was no detectable expression of ventricular myosin heavy chain (beta-subunit), suggesting that the genes for the myosin heavy chains and light chains are not expressed coordinately.     

Transitions in cardiac isomyosin expression during differentiation of the embryonic chick heart

The expression of different isoforms of the contractile protein myosin plays a major role in determining contractile characteristics in both cardiac and skeletal muscle in the adult. There is little evidence pertaining to putative changes in myosin phenotype during cardiac embryogenesis or if such changes could play a role in modulating the contractile characteristics of the developing heart. We examined isomyosin expression during cardiogenesis in the chick by indirect immunofluorescence microscopy with monoclonal antibodies to adult ventricular and atrial myosin heavy chains. Antibody specificity was characterized in the adult on the basis of immunofluorescence localization, ELISA, and protein blot immunoassay. Results show that the early embryonic chick heart has a different myosin phenotype than the later embryonic or adult heart. Both the embryonic ventricular and atrial myocardia initially expressed a myosin heavy chain that was recognized by antibody specific (in the adult) for ventricular myosin heavy chain. The ventricles remained reactive throughout life with the ventricular antibody, but reactivity of the atrial myocardium was confined to the initial 6 days of embryonic development. On the other hand, reactivity of the embryonic heart with multiple antibodies specific (in the adult) for atrial myosin was confined to the atrial myocardium throughout development. Thus, the distribution of myosin isoforms became similar to that of the adult myocardium by the time the embryonic heart achieved a 4-chambered configuration at 6 days in ovo.     

Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.     

Myosin light chain compositions of the interatrial and interventricular septa of sheep heart

Summary The myosin light chain composition of sheep interatrial and interventricular septa were analysed by one- and two-dimensional polyacrylamide gel. The interventricular septum has myosin light chain composition indistinguishable from that of ventricular myosin. Myosin from the interatrial septum contains three light chains, two of which co-migrated with the two atrial light chains (ALC₁ and ALC₂), while the third co-migrated with ventricular light chain 2 (VLC₂). ALC₁ are more abundant than ALC₂ or VLC₂ suggesting a mixed myosin population. Myosin with ALC₁ and VLC₂ light chain composition may be present, and its possible relationship with cardiac conducting cells is discussed.     

Myosin P-light chain isoenzymes in the human heart: evidence for diphosphorylation of the atrial P-LC form

Summary We studied myosin light chains (LC) of human atrium and ventricle of normal and diseased individuals by a high-resolution 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique. Atrial LCs (ALC-1, ALC-2 (=P-LC)) revealed both higher molecular weights and lower isoelectric points (IEP) than their ventricular counterparts (VLC-1, VLC-2 (=P-LC)). Different P-LC forms with their distinct myosin isoenzymes have been designated as P-LC-polymorphism and myosin P-LC isoenzymes, respectively. In the dephosphorylated state two VLC-2 forms (VLC-2 and VLC-2*) with the same MW and different IEP, but only one ALC-2 form, were found. In the partially phosphorylated state ALC-2 appeared to be single- and double-phosphorylated (three spots in the 2D-PAGE), whereas the two VLC-2 forms appeared to be single-phosphorylated each (four spots in the 2D-PAGE). Phosphoryl-transfer from ATP to the P-LC forms was studied using skinned fibers incubated with MLCK (myosin light chain kinase) and (-³²P)ATP. Ventricular myosin P-LC isoenzyme pattern was usually the same in normal and diseased patients: the VLC-2 to VLC-2* ratio was approx. 70/30, but in one patient with valvular heart disease (VHD) the relation was 55/45 (shift to the VLC-2* form). In hypertrophied atria of VHD patients a shift of the myosin P-LC isoenzyme pattern to the VLC-2* form occurred, too.     

Hormonal influences on cardiac myosin ATPase activity and myosin isoenzyme distribution

It has been recognized for a long time that changes in hormone secretion can influence cardiac function; however, the biochemical basis for these changes has only recently been clarified. In this review the influences of hormonal status on the contractile protein myosin is discussed. Myosin has a rod-like portion and a globular head and consists of two myosin heavy chains (MHC) and four light chains (LC), two of which are identical. The globular head is the site of an ATP-splitting enzyme, the myosin ATPase, and increases in myosin ATPase activity are closely related to an increased velocity of contraction of the heart. Myosin ATPase activity shows marked response to alterations in thyroid hormone, insulin, glucocorticoid, testosterone and catecholamine levels, but marked animal species differences in this response occur. Thyroid hormone administration to normal rabbits, for example, increases myosin ATPase activity markedly, but the myosin ATPase activity of hyperthyroid rats remains unchanged. In contrast, in hypothyroid rats myosin ATPase activity is markedly decreased but the hypothyroid rabbit shows no such response. These species-related differences in the hormonal response of myosin ATPase activity result from the predominance pattern of specific myosin isoenzymes. In the normal rat heart three myosin isoenzymes, v₁, V₂ and V₃, can be separated electrophoretically. Myosin V₁ predominates (70% of total myosin), and has the highest myosin ATPase activity, whereas in rabbits myosin v₃, which has a lower myosin ATPase activity, is the predominant isomyosin. Thyroid hormone administration to rabbits induces myosin V₁ predominance and therefore increases myosin ATPase activity, whereas in hyperthyroid rats only a small further increase in V₁ predominance can occur. The alterations in myosin isoenzyme predominance and myosin ATPase activity are closely correlated to changes in cardiac contractility. Hormone-induced alterations in myosin isoenzyme predominance are mediated through changes in the formation of two isoforms of myosin heavy chain. Changes in the expression of different myosin heavy chain genes are most likely responsible for the thyroid hormone and insulin-induced alterations in myosin isoenzyme predominance. Investigation of the control of myosin heavy chain formation can provide further insights into the hormonal control of a multigene family as well as broaden our understanding of the molecular events which result in altered cardiac contractility. It is currently unclear if androgens, glucocorticoids and catecholamines influence myosin ATPase activity through changes in myosin isoenzyme predominance resulting from alterations in myosin heavy chain gene expression. Post-translational modifications of myosin heavy chain and light chain polypeptides have also to be considered.     

Light Chains of Myosins from White, Red, and Cardiac Muscles

Purified preparations of rabbit skeletal white, red, and cardiac muscle myosin (WM, RM, and CM) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences in both the molecular weights and number of light chains in these myosins were found. WM has three distinct light-chain components (LC(1W), LC(2W), LC(3W)) having molecular weights of 25,500, 17,400, and 15,100, respectively. No component with a molecular weight around 15,000 is present in RM or CM. RM and CM contain components of identical molecular weights close to 25,000 and 17,000 (LC(1CR) and LC(2CR)) which, however, clearly differ in molecular weight from the corresponding subunits in WM. RM has an additional component (LC(1R)) having a slightly higher molecular weight than LC(1W) and LC(1CR). Thus differences and similarities in many biochemical properties between WM, RM, and CM, which have been described earlier, are also reflected in the light-chain components. The present results support the hypothesis that different sets of genes are active in producing components of myosin that make up different isozymic forms characteristic of each muscle type.