Myosin heavy chain isoforms in the myocardium of the atrioventricular junction of Scyliorhinus canicula (Chondrichthyes, Carcharhiniformes).

The atrioventricular junction of the fish heart, namely the segment interposed between the single atrium and the single ventricle, has been studied anatomically and histologically in several chondrichthyan and teleost species. Nonetheless, knowledge about myosin heavy chain (MyHC) in the atrioventricular myocardium remains scarce. The present report is the first one to provide data on the MyHC isoform distribution in the myocardium of the atrioventricular junction in chondrichthyans, specifically in the lesser spotted dogfish, Scyliorhinus canicula, a shark species whose heart reflects the primitive cardiac anatomical design in gnathostomes. Hearts from five dogfish were examined using histochemical and immunohistochemical techniques. The anti-MyHC A4.1025 antibody was used to detect differences in the occurrence of MyHC isoforms in the dogfish, as the fast-twitch isoforms MYH2 and MYH6 have a higher affinity for this antibody than the slow-twitch isoforms MYH7 and MYH7B. The histochemical findings show that myocardium of the atrioventricular junction connects the trabeculated myocardium of the atrium with the trabeculated layer of the ventricular myocardium. The immunohistochemical results indicate that the distribution of MyHC isoforms in the atrioventricular junction is not homogeneous. The atrial portion of the atrioventricular myocardium shows a positive reactivity against the A4.1025 antibody similar to that of the atrial myocardium. In contrast, the ventricular portion of the atrioventricular junction is not labelled, as is the case with the ventricular myocardium. This dual condition suggests that the myocardium of the atrioventricular junction has two contraction patterns: the myocardium of the atrial portion contracts in line with the atrial myocardium, whereas that of the ventricular portion follows the contraction pattern of the ventricular myocardium. Thus, the transition of the contraction wave from the atrium to the ventricle may be established in the atrioventricular segment because of its heterogeneous MyHC isoform distribution. The findings support the hypothesis that a distinct MyHC isoform distribution in the atrioventricular myocardium enables a synchronous contraction of inflow and outflow cardiac segments in vertebrates lacking a specialized cardiac conduction system.

High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload.

BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain ß (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.

Sex-related differences in muscle size explained by amplitudes of higher-threshold motor unit action potentials and muscle fibre typing.

AIM: To investigate the relationships between motor unit action potential amplitudes (MUAPAMP ), muscle cross-sectional area (mCSA) and composition (mEI), per cent myosin heavy chain (%MHC) areas and sex in the vastus lateralis (VL). METHODS: Ten males and 10 females performed a submaximal isometric trapezoid muscle action that included a linearly increasing, steady torque at 40% maximal voluntary contraction, and linearly decreasing segments. Surface electromyographic decomposition techniques were utilized to determine MUAPAMPS in relation to recruitment thresholds (RT). Ultrasound images were taken to quantify muscle mCSA and mEI. Muscle biopsies were collected to calculate %MHC areas. Y-intercepts and slopes were calculated for the MUAPAMP vs RT relationships for each subject. Independent-samples t tests and ANOVA models examined sex-related differences in mCSA, mEI, slopes and y-intercepts for the MUAPAMP vs RT relationships and %MHC areas. Correlations were performed among type IIA and total type II %MHC area, mCSA and the slopes and y-intercepts for the MUAPAMP vs RT relationships. RESULTS: Males exhibited greater slopes for the MUAPAMP vs RT relationships (P = .003), mCSA (P < .001) and type IIA %MHC (P = .011), whereas females had greater type I %MHC area (P = .010) and mEI (P = .024). The mCSA, type IIA and total II %MHC area variables were correlated (P < .001-.015, r = .596-.836) with the slopes from the MUAPAMP vs RT relationships. CONCLUSION: Sex-related differences in mCSA and MUAPAMPS of the higher-threshold MUs were likely the result of larger muscle fibres expressing type II characteristics for males.

Technical note: Protocol for electrophoretic separation of bovine myosin heavy chain isoforms and comparison to immunohistochemistry analysis.

Myosin heavy chain (MyHC) isoform composition is a primary determinant of contractile speed of muscle fibers. Currently, bovine MyHC isoforms are evaluated using time-consuming histochemical analysis by immunflourescence or ATPase activity. Electrophoretic separation of MyHC isoforms is more rapid; however, a reliable procedure without use of gradients has not been validated for cattle. Therefore, our objectives were to develop and validate a procedure for separating bovine MyHC isoforms (I, IIa, and IIx) using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and compare results to immunohistochemistry (IHC) analysis. Muscle samples were collected from masseter, sternomandibularis, diaphragm, longissimus lumborum, and cutaneous trunci within 1.5 h postmortem. To determine appropriate conditions for electrophoretic separation, several parameters of gel composition were varied. Bovine MyHC isoforms were clearly separated by increasing glycerol content of polyacrylamide gels to 37%. Identity of MyHC isoforms was confirmed using western blotting. Percent MyHC composition evaluated by gel electrophoresis was consistent with IHC (P > 0.2). Thus, SDS-PAGE produces clear separation of MyHC isoforms, and is a viable alternative to IHC-based methods.

Proteomics and immunohistochemistry identify the expression of α-cardiac myosin heavy chain in the jaw-closing muscles of sooty mangabeys (order Primates).

OBJECTIVE: The jaw-closing muscles of humans and nonprimate mammals express alpha-cardiac fibers but MyHC α-cardiac has not been identified in the jaw adductors of nonhuman primates. We determined whether MyHC α-cardiac is expressed in the superficial masseter and temporalis muscles of the sooty mangabey (Cercocebus atys), an African Old World monkey that specializes on hard seeds. DESIGN: LC-MS/MS based proteomics was used to identify the presence of MyHC Iα. Immunohistochemistry was used to analyze the composition and distribution of fiber types in the superficial masseter and temporalis muscles of eight C. atys. Serial sections were stained against MyHC α-cardiac (MYH6), as well as MyHC-1 (NOQ7.5.4D), MyHC-2 (MY-32), and MyHC-M (2F4). RESULTS: Proteomics analysis identified the presence of Myosin-6 (MyHC α-cardiac) in both heart atrium and superficial masseter. MyHC α-cardiac was expressed in abundance in the superficial masseter and temporalis muscles of all eight individuals and hybrid fibers were common. CONCLUSIONS: The identification of MyHC α-cardiac in the jaw adductors of sooty mangabeys is a novel finding for nonhuman primates. The abundance of MyHC α-cardiac indicates a fatigue-resistant fiber population characterized by intermediate speed of contraction between pure MyHC-1 and MyHC-2 isoforms. We suggest that α-cardiac fibers may be advantageous to sooty mangabeys, whose feeding behavior includes frequent crushing of relatively large, hard seeds during the power stroke of ingestion. Additional studies comparing jaw-adductor fiber phenotype of hard-object feeding primates and other mammals are needed to explore this relationship further.

Identification of targeting peptides for the diagnosis of myocarditis.

AIM: Current diagnostic tests for myocarditis are invasive and have low diagnostic value. Our aim was to identify potential targeting peptides to detect early myocarditis following intravenous delivery. MATERIALS & METHODS: We used an animal model of experimental autoimmune myocarditis and a phage display library to identify potential targeting peptides. After several steps, we selected two peptides, MyH-PhD-05 and MyH-PhD-120, for in vivo screening using fluorescent imaging. Immunofluorescence and proteonomic analysis was used to identify potential cellular and molecular targets of MyH-PhD-05. Echocardiography was used to assess functional changes. RESULTS: Peptide MyH-PhD-05 was able to detect animals with severe myocarditis even in the absence of functional changes. Immunofluorescence demonstrated that MyH-PhD-05 colocalizes with CD4+ T cells and monocytes (CD11b+) in cardiac infiltrates. CONCLUSION: We identified potential targeting peptides for the diagnosis of myocarditis. Future studies will focus on better identification of potential targets and translating this technology to clinically relevant imaging modalities.

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry.

Myosin heavy chain (MHC), the major component of the myosin motor molecule, plays an essential role in force production during muscle contraction. However, a comprehensive analysis of MHC proteoforms arising from sequence variations and post-translational modifications (PTMs) remains challenging due to the difficulties in purifying MHC (∼223 kDa) and achieving complete sequence coverage. Herein, we have established a strategy to effectively purify and comprehensively characterize MHC from heart tissue by combining size-exclusion chromatography (SEC) and middle-down mass spectrometry (MS). First, we have developed a MS-compatible SEC method for purifying MHC from heart tissue with high efficiency. Next, we have optimized the Glu-C, Asp-N, and trypsin limited digestion conditions for middle-down MS. Subsequently, we have applied this strategy with optimized conditions to comprehensively characterize human MHC and identified ß-MHC as the predominant isoform in human left ventricular tissue. Full sequence coverage based on highly accurate mass measurements has been achieved using middle-down MS combining 1 Glu-C, 1 Asp-N, and 1 trypsin digestion. Three different PTMs: acetylation, methylation, and trimethylation were identified in human ß-MHC and the corresponding sites were localized to the N-terminal Gly, Lys34, and Lys129, respectively, by electron capture dissociation (ECD). Taken together, we have demonstrated this strategy is highly efficient for purification and characterization of MHC, which can be further applied to studies of the role of MHC proteoforms in muscle-related diseases. We also envision that this integrated SEC/middle-down MS strategy can be extended for the characterization of other large proteins over 200 kDa.

Electrophoretic separation of reptilian skeletal and cardiac muscle myosin heavy chain isoforms: dependence on gel format.

This report provides a comparison of multiple gel formats to study myosin heavy chain (MHC) isoforms that are expressed in reptilian skeletal and cardiac muscles of five turtle species, water monitor, and prehensile tailed skink. Three gel formats were tested. The results identify one format that is superior, for the overall extent of electrophoretic separation and for the assessment of the number of MHC isoforms in reptilian striated muscles. The same format was shown previously to separate MHC isoforms that are expressed in American alligator. The results also show that another gel format reveals the distinct electrophoretic mobility of MHC isoforms in atrial, ventricular, and jaw adductor samples, compared to those expressed in skeletal muscles in the limbs and elsewhere in the body. In addition, the results reveal that the electrophoretic mobility of specific MHC isoforms, relative to other isoforms, depends on the gel format, as shown previously for mammalian and avian species. The discovery of the expression of masticatory MHC, which is abundantly expressed in jaw adductors of members of Carnivora and several other vertebrate orders, in the homologous muscles of prehensile tailed skink, an herbivore, and the carnivorous water monitor, was made during the course of this study.

Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a.

Type V myosins constitute the main cargo-transporting class of myosin motors in higher eukaryotes. They are mainly defined by their C-terminal globular domain, which is required for cargo binding as well as for motor auto-inhibition in the absence of cargo. To date, high-resolution structures only exist for globular domains from yeast. Since the majority of cellular cargoes in yeast are very different from the cargoes in higher eukaryotes, structural insights into the domain organization of globular domains from human type V myosins are important. The globular domain of human Myo5a was cloned, expressed and crystallized and data sets were collected. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.04, b = 86.70, c = 131.41 Å, α = ß = γ = 90°, and diffracted with data-collection quality to 2.5 Šresolution.

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry.

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS-PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA-F8 (anti-MHC slow/I), SC-71 (anti-MHC 2a and 2x), 10F5 (anti-MHC 2b), and BF-35 (anti-MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS-PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS-PAGE technique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.

Electrophoretic separation of myosin heavy chain isoforms using a modified mini gel system.

The electrophoretic separation of myosin heavy chain isoforms from muscle biopsy homogenates has been widely practiced in the field of exercise physiology to examine how intrinsic (i.e., aging) and extrinsic (i.e., training) factors affect muscle phenotype. In the past, various research groups have used large and mini polyacrylamide gel systems to perform this delicate methodology. As technology has progressed, additional gel formats have been introduced, but available methodologies appear to be lacking. In this investigation, we successfully separated 3 distinct myosin heavy chain isoforms from various muscle samples using a modified mini gel system that can load up to 26 samples per gel. This article will outline our allocated protocol and discuss potential troubleshooting considerations for other researchers performing this intricate methodology. The outlined methodology has resulted in an ability to clearly resolute 3 distinct bands at molecular weights attributed to the myosin heavy chain isoforms in human skeletal muscle at a wide range of human ages (20-78 years). As additional technologies become available, the need to modify and adapt existing electrophoretic protocols for myosin heavy chain isoform separation and other protocols will continue to be evident.

Electrophoretic mobility of cardiac myosin heavy chain isoforms revisited: application of MALDI TOF/TOF analysis.

The expression of two cardiac myosin heavy chain (MyHC) isoforms in response to the thyroid status was studied in left ventricles (LVs) of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCß by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCß isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCß isoforms analyzed by the SDS-PAGE.

Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle.

In this short communication we describe a specific protocol for SDS-PAGE separation of adult bovine myosin heavy-chain (MyHC) isoforms. The conditions defined in this protocol allow a good separation with a good reproducibility of the four MyHC isoforms (MyHC I, IIa, IIx, IIb) identified in adult skeletal muscle of this species. This procedure uses mini-gel electrophoresis system and does not involve preparation of gradient separating gels. In addition, this protocol can also be applied to the electrophoretic separation of ovine and camel MyHC isoforms.

Differential muscular myosin heavy chain expression of the pectoral and pelvic girdles during early growth in the king penguin (Aptenodytes patagonicus) chick.

Continuous growth, associated with a steady parental food supply, is a general pattern in offspring development. So that young chicks can acquire their locomotor independence, this period is usually marked by a fast maturation of muscles, during which different myosin heavy chain (MyHC) isoforms are expressed. However, parental food provisioning may fluctuate seasonally, and offspring therefore face a challenge to ensure the necessary maturation of their tissues when energy is limited. To address this trade-off we investigated muscle maturation in both the pectoral and pelvic girdles of king penguin chicks. This species has an exceptionally long rearing period (1 year), which is prolonged when parental food provisioning is drastically reduced during the sub-Antarctic winter. Approximately 1 month post hatching, chicks acquire a functional pedestrian locomotion, which uses pelvic muscles, whereas swimming, which uses the pectoral muscles, only occurs 1 year later. We therefore tested the hypothesis that the MyHC content of the leg muscles reaches a mature state before those of the pectoral muscles. We found that leg muscle MyHC composition changed with the progressive acquisition of pedestrian locomotion, whereas pectoral muscle fibres reached their mature MyHC profile as early as hatching. Contrary to our predictions, the acquisition of the adult profile in pectoral muscles could be related to an early maturation of the contractile muscular proteins, presumably associated with early thermoregulatory capacities of chicks, necessary for survival in their cold environment. This differential maturation appears to reconcile both the locomotor and environmental constraints of king penguin chicks during growth.

Detailed tuning of structure and intramolecular communication are dispensable for processive motion of myosin VI.

Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.

Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus.

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested-actin, myosin heavy and light chain, and IQGAP-as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis.

Mapping of O-linked beta-N-acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle.

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O-GlcNAc modified. Moreover, it was demonstrated that O-GlcNAc moieties involved in contractile protein interactions could modulate Ca(2+) activation parameters of contraction. In order to better understand how O-GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O-GlcNAc modification in purified contractile protein homogenates. Using an MS-based method that relies on mild beta-elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O-GlcNAc site in the subdomain four of actin and four O-GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O-GlcNAc sites might modulate the actin-tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein-protein interactions but could also modulate the contractile properties of skeletal muscle.

GelBandFitter--a computer program for analysis of closely spaced electrophoretic and immunoblotted bands.

GelBandFitter is a computer program that uses non-linear regression techniques to fit mathematical functions to densitometry profiles of protein gels. This allows for improved quantification of gels with partially overlapping and potentially asymmetric protein bands. The program can also be used to analyze immunoblots with closely spaced bands. GelBandFitter was developed in Matlab and the source code and/or a Windows executable file can be downloaded at no cost to academic users from http://www.gelbandfitter.org.

Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle.

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51+/-0.04) than phasic muscle myosin (-0.15+/-0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092+/-0.022 pN for phasic muscle and 0.084+/-0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.