Cardiac Muscle Myosin Research Articles

Light Chain Kinase Specificity in Cardiac Myosin

Human ventricular cardiac myosin regulatory light chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain Ser15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (Ser20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine ventricular myosin, and chicken gizzard myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.

3-Dimensional Structure of Human Cardiac Muscle Myosin Filaments by Electron Microscopy and Single Particle Analysis

In all muscles the interaction between actin and myosin filaments leads to muscle contraction and force production mediated by the hydrolysis of ATP. Actin filament structure is well understood to high resolution, but myosin filament structure is much less well defined. Myosin filaments are formed from complicated arrangements of myosin molecules and accessory proteins (e.g. C-protein [MyBP-C] and titin). We compare the arrangement of the myosin heads in the different species and how these arrangements change in diseased muscle. We have developed a method to solve the 3-dimensional (3D) structure of myosin filaments by single particle analysis of electron microscope (EM) data. We already defined the 3D structure of myosin filaments of various muscles from different species by both X-ray diffraction modelling and EM and single particle analysis including insect flight muscle, scallop striated muscle, fish skeletal muscle and rabbit cardiac muscle. We are now studying the 3D structure of myosin filaments isolated from human heart muscles.Mutations in cardiac myosin, C-protein and titin are known to be associated with cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand myosin-associated heart disease, it is important to understand the 3D structure of myosin filaments in normal heart muscle.Recently we have developed procedures to isolate human cardiac muscle myosin filaments preserving their highly ordered pseudo-helical structure thus making them amenable, for the first time, to EM and single particle image analysis. We have collected EM data from myosin filaments isolated from both normal and failing hearts, and have so far processed the data from normal heart muscle. Analysis of the 3D structure of myosin filaments in normal heart muscle will permit the structural effects of known myosin filaments-associated mutations to be investigated in detail.

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten Vmax and KM for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.

Differential Effects of Alpha Vs Beta Myosin Heavy Chain on the Kinetcs and Mechanics of Familial Hypertrophic Cardiomyopathy Mutations in the Myosin Regulatory Light Chain

Cardiac muscle myosin is comprised of two heavy chains (MHCs), two essential light chains, and two regulatory light chains (RLCs). The MHC contains both the ATPase and actin binding domains. It has been shown that the contractile properties of myosin can be tuned by the MHC isoform and that the MHC isoform distribution in the human heart changes during heart failure from predominantly beta isoform to all beta. One cause of heart failure is familial hypertrophic cardiomyopathy (FHC) which is triggered by mutation of sarcomeric proteins including the RLC. Although the RLC is spatially separated from the myosin active site, it appears to have a role in tuning myosin kinetics. In order to examine how two RLC mutations implicated in FHC, N47K and R58Q, affect the kinetic and mechanical properties of beta isoform myosin, we exchanged porcine cardiac RLC with recombinant mutant RLC. We examined the contractile properties of these mutants using the in vitro motility assay and compared these results to our earlier results with mutant RLCs on the alpha -MHC background. Regardless of MHC isoform, the mutations cause reductions in force and power output. However, on the alpha MHC backbone, R58Q shows differences in calcium handling and an elevated ATPase rate which is not seen on the beta backbone. Also, both mutants show increases in duty cycle on the alpha MHC but not the beta. These data suggest that small changes in the myosin structure, far from the active site, can disrupt the contractile properties of the motor depending on the MHC isoform.

Muscle myosin filaments: cores, crowns and couplings.

Myosin filaments in muscle, carrying the ATPase myosin heads that interact with actin filaments to produce force and movement, come in multiple varieties depending on species and functional need, but most are based on a common structural theme. The now successful journeys to solve the ultrastructures of many of these myosin filaments, at least at modest resolution, have not been without their false starts and erroneous sidetracks, but the picture now emerging is of both diversity in the rotational symmetries of different filaments and a degree of commonality in the way the myosin heads are organised in resting muscle. Some of the remaining differences may be associated with how the muscle is regulated. Several proteins in cardiac muscle myosin filaments can carry mutations associated with heart disease, so the elucidation of myosin filament structure to understand the effects of these mutations has a clear and topical clinical relevance.

Prenatal exposure to maternal low protein diet suppresses replicative potential of myocardial cells

Background and aims We have previously shown that a maternal low protein (LP) diet during pregnancy results in severe depression of neonatal heart contractility due, in part, to an increase in apoptotic loss of cardiomyocytes. The aim of this study was to examine if maternal LP diet would alter replicative potential of neonatal myocardial cells. Methods and results We determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on relative numbers of mitotic myocardial cells in male offspring at birth and at 7–28 days post-partum. Myocardial cells undergoing mitosis were identified by dual-immunofluorescence of cardiac sections for cardiac muscle myosin and phosphorylated histone 3, whereas cells within the cell cycle were identified by immunoreactivity for Ki67 at 14–28 days post-partum. Neonates from control dams displayed the expected gradual decline in mitotic cells from birth to 28 days post-partum. Hearts from LP offspring had lower numbers of mitotic cells at birth, compared to controls, suggestive of subnormal muscle cell numbers at that stage. When placed in normal diet, LP offspring developed increased myocardial mitosis at 7 days compared to controls, which normalized to control levels at 21–28 days post-partum. An increase in Ki67-positive myocardial cells was also observed in the LP exposed group at 28 days of age. Conclusion Maternal LP diet suppresses myocardial replicative potential and this likely contributes to reduced cell numbers at birth. This suppression is lifted by a protein-replete diet which stimulates post-natal replication of myocardial cells and likely results in a catching-up in cell numbers.

Myosin filament 3D structure in mammalian cardiac muscle

A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac).

Three-dimensional structure of vertebrate cardiac muscle myosin filaments.

Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases.

Replacement of Nonmuscle Myosin II-B with II-A Rescues Brain but Not Cardiac Defects in Mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B(-)/B(-) mice, most B(a*)/B(a*) mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B(-)/B(-) mice, 15 B(a*)/B(a*) mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the B(a*)/B(a*) mice showed evidence for hydrocephalus that is always found in B(-)/B(-) mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in B(a*)/B(a*) mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.

Особливості функціонування міозинової АТРази серцевого м'яза

Особливості функціонування міозинової АТРази серцевого м'яза

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions.

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.

Truncation of Vertebrate Striated Muscle Myosin Light Chains Disturbs Calcium-Induced Structural Transitions in Synthetic Myosin Filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca2+-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca2+-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca2+-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.

Calcium-Induced Structural Changes in Synthetic Myosin Filaments of Vertebrate Striated Muscles

Using negative staining, freeze-drying, and shadowing techniques in electron microscopy we have for the first time demonstrated Ca-induced reversible structural transitions in the synthetic filaments of dephosphorylated column-purified rabbit skeletal and cardiac muscle myosins formed by dialysis against solutions containing 120 mM KCl, 1 mM MgCl2, 10 mM imidazole–HCl buffer (pH 7.0), and either 0.1 mM CaCl2 or 1 mM EGTA. It has been revealed that the compact ordered structure of the filaments with myosin heads and subfragments-2 (S2) disposed close to the filament backbone with an axial periodicity of about 14.5 nm in the absence of Ca2+ transforms into a spread disordered structure due to the movement of the heads and S2 away from the filament surface in the presence of Ca2+. Increasing the pH from neutrality to pH 7.8 leads to a spread, disordered structure while decreasing the pH value to 6.5 returns the filaments to their compact, rather ordered state independent of the Ca2+ concentrations used. The fact that the reversible structural transitions in synthetic filaments of myosin are observed in the absence of actin and actin- and myosin-associated proteins suggests that Ca2+-induced S2 movement is an intrinsic property of myosin itself. Ca2+-induced S2 mobility may reflect the existence of functionally significant communications between the myosin head domains and the tails of myosin molecules in thick filaments, and its disappearance can be an indicator of the impairment of these communications, for example, in acute ischemia and myocardial infarction.

Yeast myosin II: a new subclass of unconventional conventional myosins?

Myosin II is the founder member of a large and structurally diverse clan of actin-based motor proteins. The native myosin II molecule is a hexamer consisting of two heavy chains, two essential light chains (ELC), and two regulatory light chains (RLC). For convenience, the myosin IIs are often subdivided into four subclasses: vertebrate skeletal and cardiac muscle myosin II form one subclass, vertebrate smooth muscle and nonmuscle myosin II a second, invertebrate muscle a third, and protozoan myosin II a fourth [Sellers and Goodson, 1995]. Different mechanisms of regulation may exist between myosins within a single subclass yet all myosin IIs share a common three-domain structure; the N-terminus of the heavy chain forms two globular heads that contain the ATP- and actin-binding sites and the alpha-helical neck region that is stabilised by the binding of the two classes of light chains, whilst the C-terminus forms an extended coiled-coil tail that can consist of anywhere between 700 and 1,200 amino acids. In nonmuscle cells, myosin II has at least two well-defined functions, cell locomotion and cytokinesis. Yeast cells do not locomote, and their mechanism of cytokinesis involves the deposition of a cross-wall or septum. However, in the fission yeast, Schizosaccharomyces pombe, deposition of the septum is anticipated by the appearance of a contractile actomyosin ring [Marks and Hyams, 1985; May et al., 1997; Kitayama et al., 1997] and actin is also present at the bud neck during cytokinesis in the budding yeast, Saccharomyces cerevisiae [Kilmartin and Adams, 1984]. Here we report a phylogenetic analysis of the N-terminal head domains of the myosin IIs from both yeasts, a structural analysis of the tail domains of these proteins and we speculate as to the nature of the light chains that regulate their function. On the basis of these findings, we propose that the yeast myosin IIs constitute a divergent fifth class of "unconventional" conventional myosins.

A Myosin-Derived Peptide C109 Binds to GLUT4-Vesicles and Inhibits the Insulin-Induced Glucose Transport Stimulation and GLUT4 Recruitment in Rat Adipocytes

The yeast-based two-hybrid screening of a human cardiac myocyte cDNA library revealed a peptide, C109 that interacted with the C-terminal cytoplasmic domain of GLUT4 (GLUT4C). cDNA-duced amino acid sequence of C109 was identical to the human cardiac muscle myosin heavy chain β isoform sequence 1469-1909. GST-fusion protein of C109 (GST-C109) bound synthetic GLUT4C-peptide in vitro,but not GLUT1C-peptide. GST-C109 avidly bound to the GLUT4-vesicles isolated from basal rat adipocytes but not those isolated from insulin treated adipocytes. Furthermore, the incorporation of C109 into rat adipocytes greatly reduced the plasma membrane GLUT4 level and the 3-O-methyl D glucose flux in host cells without affecting total cellular GLUT4 content. These findings suggest that myosin or a myosin-like protein plays a key role in insulin-regulated movement of GLUT4 to the plasma membrane in rat adipocytes.

Amino Acid Composition and Nτ-Methylhistidine Contents of Bovine and Porcine Cardiac Muscle Tissues

The actin and collagen contents of adult bovine and porcine cardiac muscle tissues have been determined from the amounts of Nτ-methylhistidine and 5-hydroxylysine found in their acid hydrolysates. Bovine and porcine cardiac αc-actins from hearts of adult cows and sows contained 375 and 376 amino acids, respectively, and 1 mol of Nτ-methylhistidine/mol of protein, 42 000 Da, consistent with the values for all eukaryotic actins. Since the molar ratio of myosin to actin in cardiac muscles is known to be 1:4, the myosin content can be estimated from the amounts of αc-actin present. αc-Actin accounts for an estimated 9.7% of total cardiac muscle mass and myosin for about 27.6−28.0% of total protein, corresponding to 16.3 and 48.6% of the total myofibrillar proteins (57.6%). Total cardiac collagen ranged from 3.9% in porcine to 4.8% in bovine hearts. Cardiac muscles have a very good balance of EAA9 ranging from 49.8 to 50.3%, compared to the FAO/WHO reference essential amino acid (EAA9) pattern value of 33.9% f...

Primary structure of chicken cardiac myosin S-1 heavy chain.

The sequence of the NH2-terminal 830 amino acid residues of chicken cardiac ventricular muscle myosin subfragment-1 (S-1) was determined. S-1 was obtained by limited chymotryptic digestion, and cleaved into three characteristics fragments (23, 41, and 22 kDa fragments) by limited tryptic digestion. These fragments were isolated by gel filtration on a Sephadex G-100 column, followed by cation-exchange chromatography on a CM-52 column and reverse-phase HPLC. The isolated fragments were sequenced completely. Peptides overlapping the 23 and 41 kDa fragments and also overlapping the 41 and 22 kDa fragments were obtained by cleaving S-1 with cyanogen bromide, and sequenced completely. We also obtained a minor fragment, the 20 kDa fragment, in addition to the three characteristic fragments. Amino acid compositions of the cyanogen bromide peptides of the 20 kDa fragment indicated that a portion of S-1 heavy chains had lost their COOH-terminal 21 residues during limited tryptic digestion. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 32, epsilon-N-trimethyllysine residues at 127 and 549, and 3-N-methylhistidine at 754.