Characterization Of Myosin Heavy Chain Research Articles

Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.

Characterization of myosin heavy chain (MYH) genes and their differential expression in white and red muscles of Chinese perch, Siniperca chuatsi

Fish skeletal muscle is composed of two anatomically and functionally different fiber layers, white or fast and red or slow muscles. Myosin, the major structural protein of fish skeletal muscle, contains multiple myosin heavy chain (MYH) isoforms involved in the high plasticity of muscle in response to varying functional demands and/or environmental changes. In this study, we comparatively assayed the cellular and ultrastructural feature of white and red skeletal muscles. Then, a total of 28 class II myosin heavy chain genes were identified in by searching the Chinese perch genome database. Among them, 14 genes code for the fast-muscle-type myosin heavy chain, and 7 genes code for the slow-muscle-type myosin heavy chain. Further, the different isoform gene structures, function domains, phylogenetic relations, and muscle-fiber type-specific expression were characterized. This is the first systematic work on the molecular characterization of class II myosin heavy chain isoforms and the differential analysis of their expression in red and white muscle tissues in Chinese perch Siniperca chuatsi. Our work provided valuable information for a better understanding of myh genes and their molecular characteristics, and the correlations of multiple myosin isoforms with potential functions in response to varying functional demands and/or environmental changes.

Molecular characterization of myosin heavy chain and its involvement in muscle growth and development in the Yesso scallop Patinopecten yessoensis

Myosin found in scallop adductor muscles is a regulatory protein that is responsible for muscle contraction. Myosin heavy chains (MyHCs) form the core component of myosin and play important roles in myofibril assembly and maintenance. In the present study, we obtained the full-length cDNA sequences encoding MyHC from the Yesso scallop Patinopecten yessoensis and investigated its expression patterns as well as regulatory functioning in muscle growth and development. Py-MyHC was 6054 bp in length and was comprised of a 5′-untranslated region (51 bp), a 3′-untranslated region (180 bp), and an open reading frame (5823 bp) encoding 1940 amino acids. Analysis of the MyHC sequences in bivalves revealed that they were closely homologous to each other, with identities ranging from 74.48% to 95.16%. qPCR analysis and immunohistochemistry staining revealed the existence of tissue-specific expression patterns for Py-MyHC with maximal expression occurring in the striated adductor muscles. Whole-mount in situ hybridization showed that during larval development, Py-MyHC mRNA was mainly expressed in the velum retractor muscles, and in the anterior and posterior adductor muscles. Furthermore, noticeable defects in myofibril structure were found in the striated adductor muscles following the application of RNA interference. Based on the above, it is considered that MyHCs play a crucial role in myofibril assembly and maintenance in both larvae and adults. These findings are considered to provide useful information with regard to the functional basis of muscle growth and muscle production with potential implications for actual aquaculture production.

Characterization of myosin heavy chains from the mantle muscles of cephalopods

Myosin heavy chain (MyHC) genes from the mantle muscles of three cephalopod species, namely, golden cuttlefish Sepia esculenta, Japanese common squid Todarodes pacificus, and spear squid Loligo bleekeri were subjected to synthesis of first strand cDNA by reverse transcription-PCR. As a result of sequencing of the obtained cDNA fragments, the MyHCs from S. esculenta and L. bleekeri were found to consist of 1936 amino acids, while T. pacificus MyHC consisted of 1939 residues. The sequences were found to be 73%, 55%, and 41% identical to those of the Ca2+-regulated scallop MyHCs, human skeletal MyHC, and chicken gizzard (smooth muscle) MyHC, respectively. The cephalopod MyHCs conserved ATP- and actin-binding sites as well as light chain binding sites in the head (S1) region with the clear differences in sequence homology from those of higher organisms, and typical ?-helical heptad repeats involved in the formation of coiled-coil structure in the rod region. The sequences of the two surface loops at the 25/50 kDa and 50/20 kDa junctions of the motor domain were found to be conserved among cephalopods compared to those from the other sources. In the phylogenetic tree based on the amino acid sequences, the cephalopod MyHCs formed a cluster, clearly different from the other invertebrate counterparts, in accordance with the results of tertiary structure modeling of the head regions.

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 β-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 β-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.

Characterization of myosin heavy chain and its gene in Amoeba proteus.

Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNAs encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, as checked by indirect immunofluorescence microscopy and by immunoelectron microscopy. The open reading frame of a cloned myosin cDNA contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While the amino-acid sequence of the globular head region of amoeba's myosin had a high degree of similarity with that of myosins from various organisms, the tail region building a coiled-coil structure did not show a significant sequence similarity. There appeared to be at least three different isoforms of myosins in amoebae, with closely related amino acids in the globular head region.

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study.

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.

Characterization of myosin heavy chain by cyanogen bromide peptide maps.

Procedures have been developed for the preparation of pure myosin heavy chain (h-myosin) by preparative gel electrophoresis, and for the characterization of h-myosin by cyanogen bromide peptide mapping. Major sources of error are the oxidation of methionine and the proteolytic splitting of the chain during purification. These errors have been eliminated. A peculiar feature is the doubling or quadrupling of a peptide of molecular weight 17 000. The results show structural differences between isomyosins derived from myonal types within the same animal, as well as interspecies differences.

<b>Characterization of myosin heavy chains in cell cultures of embryonic chicken breast </b><b>muscle </b>

<b>Characterization of myosin heavy chains in cell cultures of embryonic chicken breast </b><b>muscle </b>