Establishment of Mouse Model of MYH9 Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies,Renal Glomerulosclerosis and Hearing Disability

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation.We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm.In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response.These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.     

Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.     

MYH9-dependent polarization of ATG9B promotes colorectal cancer metastasis by accelerating focal adhesion assembly

Tumour metastasis is a major reason accounting for the poor prognosis of colorectal cancer (CRC), and the discovery of targets in the primary tumours that can predict the risk of CRC metastasis is now urgently needed. In this study, we identified autophagy-related protein 9B (ATG9B) as a key potential target gene for CRC metastasis. High expression of ATG9B in tumour significantly increased the risk of metastasis and poor prognosis of CRC. Mechanistically, we further find that ATG9B promoted CRC invasion mainly through autophagy-independent manner. MYH9 is the pivotal interacting protein for ATG9B functioning, which directly binds to cytoplasmic peptide segments aa368–411 of ATG9B by its head domain. Furthermore, the combination of ATG9B and MYH9 enhance the stability of each other by decreasing their binding to E3 ubiquitin ligase STUB1, therefore preventing them from ubiquitin-mediated degradation, which further amplified the effect of ATG9B and MYH9 in CRC cells. During CRC cell invasion, ATG9B is transported to the cell edge with the assistance of MYH9 and accelerates focal adhesion (FA) assembly through mediating the interaction of endocytosed integrin β1 and Talin-1, which facilitated to integrin β1 activation. Clinically, upregulated expression of ATG9B in human CRC tissue is always accompanied with highly elevated expression of MYH9 and associated with advanced CRC stage and poor prognosis. Taken together, this study highlighted the important role of ATG9B in CRC metastasis by promoting focal adhesion assembly, and ATG9B together with MYH9 can provide a pair of potential therapeutic targets for preventing CRC progression.Subject terms: Metastasis, Oncogenes     

Podocyte-specific deletion of Myh9 encoding nonmuscle myosin heavy chain 2A predisposes mice to glomerulopathy

Genome-wide association studies linked single-nucleotide polymorphisms (SNPs) at the MYH9 locus to chronic kidney disease among African-Americans, particularly glomerular diseases such as HIV nephropathy and idiopathic focal and segmental glomerulosclerosis (FSGS). However, these MYH9 SNPs are intronic, and despite extensive sequencing, a causal variant remains elusive. To investigate the role of MYH9 in kidney disease, we selectively deleted Myh9 from mouse podocytes and found that mutant C57BL/6 mice did not develop renal insufficiency or proteinuria compared to control littermates, even when the mice were aged for 9 months. To explain the surprisingly normal phenotype, we considered genetic redundancy with the paralog Myh10 in podocytes, but we found that Myh10 was not expressed in podocytes in Myh9-deficient or control mice. We tested whether Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by doxorubicin hydrochloride (Adriamycin), and we found that Myh9 podocyte-deleted mice developed proteinuria and glomerulosclerosis, while control mice were resistant. In summary, Myh9 podocyte deletion in C57BL/6 mice results in susceptibility to experimental doxorubicin hydrochloride glomerulopathy. We review evidence that MYH9 dysfunction in humans results in similar susceptibility and place our data, the first examination of Myh9 kidney disease in experimental animals, in the context of recent findings in human kidney disease, including the role of APOL1.     

肌球蛋白重链基因在人类遗传性疾病中的研究进展

肌球蛋白超家族通过水解ATP,将化学能转化为机械能,在细胞迁移、肌肉收缩等多种生理活动中发挥重要的作用。其中,肌球蛋白Ⅱ类分子是肌细胞和非肌细胞中肌丝的重要组成成分。一个完整的肌球蛋白Ⅱ类分子是由2条肌球蛋白重链(myosin heavy chain, MyHC)和2对不同的轻链组成的六聚体。在人体中,存在多种MyHC亚型,分别由不同的MYH基因家族成员编码。迄今为止,人们已经发现MYH基因家族中多个成员的不同突变与人类遗传性疾病相关。其中,MYH2突变可以导致一类以眼肌麻痹为主要特征的骨骼肌疾病;MYH3和MYH8突变可以引起远端关节挛缩综合征;MYH7突变即可以引起骨骼肌疾病包括肌球蛋白沉积性肌病和Laing远端肌病,也与肥厚性心肌病的发生密切相关;MYH9突变可以导致一类以巨大血小板、血小板减少和中性粒细胞包涵体为特征的MYH9相关性疾病。本文简要介绍MYH基因的表达特点,着重阐述MYH基因与人类遗传性疾病之间的相关性及研究进展。     

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.     

Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9-Rad1-Hus1

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.     

Haploinsuffciency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy

Myotonic dystrophy type 2 is caused by a (CCTG)/(CCUG)n repeat expansion in the first intron of the ZNF9 gene. The pathomechanism for the myotonic dystrophies is not well understood and the role of ZNF9 in myotonic dystrophy type 2 pathogenesis has not been fully clarified. We characterized Znf9+/- mice, in which the expression of Znf9 was significantly decreased, and found that their phenotype reflects many of the features of myotonic dystrophy, including muscle histological morphology, and myotonic discharges and heart conduction abnormalities, shown by electromyography and electrocardiogram analysis, respectively. Znf9 is normally highly expressed in heart and skeletal muscle, where skeletal muscle chloride channel 1 (Clc1) plays an important role. Clc1 expression was dramatically decreased in Znf9+/- mice. Znf9 transgenic mice raised Znf9 and Clc1 expression and rescued the myotonic dystrophy phenotype in Znf9+/- mice. Our results suggest that the Znf9 haploinsufficiency contributes to the myotonic dystrophy phenotype in Znf9+/- mice.     

Prognostic Significance of MYH9 Expression in Resected Non-Small Cell Lung Cancer

Introduction

Myosin-9 (MYH9) belongs to the myosin superfamily of actin-binding motor protein. Recently, MYH9 has been thought to be associated with cancer cell migration, invasion, and metastasis. The aims of this study were to immunohistochemically examine MYH9 expression in surgically resected non-small cell lung cancer (NSCLC), and evaluate its correlations with clinicopathological parameters and the prognosis of patients.

Methods

MYH9 expression was immunohistochemically studied in 266 consecutive resected NSCLCs, and its associations with clinicopathological parameters were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of MYH9 expression on survival.

Results

MYH9 expression was detected in 102 of 266 (38.3%) NSCLCs. MYH9 expression was significantly correlated with the adenocarcinoma histology (P = 0.014), poorer differentiation ((P = 0.033), intratumoral vascular invasion and lymphatic invasion ((P = 0.013 and P = 0.045 respectively), and a poorer prognosis ((P = 0.032). In addition, multivariable analysis revealed that MYH9 expression independently predicted a poorer survival (HR, 2.15; 95%CI, 1.17-3.92; (P = 0.01).

Conclusion

The present study revealed that MYH9 is expressed in a subset of NSCLC with a more malignant nature, and its expression is an indicator of a poorer survival probability.     

Mice lacking the gene encoding for MMP-9 and resistance artery reactivity

OBJECTIVES: To define the link between the deletion of gene encoding for metalloproteinase 9 and resistance artery reactivity, we studied in vitro smooth muscle and endothelial cell function in response to pressure, shear stress, and pharmacological agents. BACKGROUND: Matrix metalloproteinases play a crucial role in the regulation of extracellular matrix turnover and structural artery wall remodeling. METHODS: Resistance arteries were isolated from mice lacking gene encoding for MMP-9 (KO) and their control (WT). Hemodynamic, pharmacology approaches, and Western blot analysis were used in this study. RESULTS: The measurement of blood pressure in vivo was similar in KO and WT mice. Pressure-induced myogenic tone, contractions to angiotensin-II and phenylephrine were similar in both groups. The inhibition of MMP2/9 ((2R)-2-[(4-biphenylylsulfonyl) amino]-3-phenylpropionic acid) significantly decreased myogenic tone in WT and had no effect in KO mice. Relaxation endothelium-dependent (flow-induced- dilation 41.3+/-0.6 vs. 21+/-1.6 at 10 microl/min in KO and WT mice, respectively, P<0.05) and eNOS expression were increased in KO compared to WT mice. The inhibition of eNOS with L-NAME significantly decreased endothelium response to shear stress, which was more pronounced in KO mice resistance arteries (-26.83+/-2.5 vs. -15.84+/-2.3 at 10 microl/min in KO and WT, respectively, P<0.05). However, the relaxation to exogenous nitric oxide-donor was similar in both groups. CONCLUSION: Our study provides evidence of a selective effect of MMP-9 on endothelium function. Thus, MMP-9 gene deletion specifically increased resistance artery dilation endothelium-dependent and eNOS expression. Based on our results, MMP-9 could be a potential therapeutic target in cardiovascular disease associated with resistance arteries dysfunction.     

A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.     

In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice

We previously reported that deletion of the Fgf2 gene (Fgf2-/-) resulted in decreased bone mass in adult mice. This study examines the effect of haplo-insuffiency (Fgf2+/-) on bone loss in vertebrae from these mutant mice. Fgf2+/+ mice attained peak bone mass at 8-9 months of age. In contrast BMD was significantly reduced in vertebrae from adult (8-9) Fgf2+/- mice. Exogenous FGF-2 rescued reduced bone nodule formation in Fgf2+/- and Fgf2-/- cultures. Runx2 mRNA was reduced in cultures from Fgf2+/- and Fgf2-/- mice. FGF receptor2 mRNA and protein were markedly reduced in Fgf2+/- and Fgf2-/- mice. Decreased bone formation in Fgf2 mutant mice may correlate with impaired FGFR signaling, decreased Runx2 gene expression.     

Transgenic expression of matrix metalloproteinase-9 causes adult-onset emphysema in mice associated with the loss of alveolar elastin

Matrix metalloproteinase (MMP)-9 has been consistently identified in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, its role in the development of the disease remains undefined. Mice that specifically express human MMP-9 in their macrophages were generated, and morphometric, biochemical, and histological analyses were conducted on the transgenic and littermate control mice over 1 yr to determine the effect of macrophage MMP-9 expression on emphysema formation and lung matrix content. Lung morphometry was normal in transgenic mice at 2 mo of age (mean linear intercept = 50+/-3 littermate mice vs. 51+/-2 transgenic mice). However, after 12 mo of age, the MMP-9 transgenic mice developed significant air space enlargement (mean linear intercept = 53+/-3 littermate mice vs. 61+/-2 MMP-9 transgenic mice; P<0.04). Lung hydroxyproline content was not significantly different between wild-type and transgenic mice, but MMP-9 did significantly decrease alveolar wall elastin at 1 yr of age (4.9+/-0.3% area of alveolar wall in the littermate mice vs. 3.3+/-0.3% area of alveolar wall in the MMP-9 mice; P<0.004). Thus these results establish a central role for MMP-9 in the pathogenesis of this disease by demonstrating that expression of this protease in macrophages can alter the extracellular matrix and induce progressive air space enlargement in mice.     

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.     

Differential effects of prenatal stress in 5-Htt deficient mice: towards molecular mechanisms of gene × environment interactions

Prenatal stress (PS) has been shown to influence the development of the fetal brain and to increase the risk for the development of psychiatric disorders in later life. Furthermore, the variation of human serotonin transporter (5-HTT, SLC6A4) gene was suggested to exert a modulating effect on the association between early life stress and the risk for depression. In the present study, we used a 5-Htt×PS paradigm to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition, anxiety- and depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt +/-) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression profiling was performed using the Affymetrix GeneChip? Mouse Genome 430 2.0 Array. 5-Htt +/- offspring showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt +/- mice to PS was associated with increased depressive-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt +/- genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype×PS manner, indicating a gene×environment interaction at the molecular level. In conclusion, our data suggest that although the 5-Htt +/- genotype shows clear adaptive capacity, 5-Htt +/- mice--particularly females--at the same time appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression profiles suggest that distinct molecular mechanisms mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction.     

Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "D?hle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.     

Identification of a novel MYH9 mutation (p. V782Y) in a Chinese patient with thrombocytopenia: a case report

MYH9-related diseases (MYH9-RD) are a group of autosomal dominant diseases caused by mutations in the MYH9 gene, which are featured by thrombocytopenia, giant platelets and granulocyte cytoplasmic inclusion bodies. MYH9-RD patients generally suffer from bleeding syndromes, progressive kidney disease, deafness, or cataracts. Here, we reported on a case of MYH9-RD. A novel heterozygous mutation of MYH9 (c.2344-2345delGTinsTA, p.T782Y) was discovered by targeted sequencing technology. Immunofluorescence analysis of neutrophils confirmed abnormal aggregation of MYH9 protein. The results of this study should expand the MYH9 gene mutation spectrum and provide reference for subsequent researchers and genetic counseling.     

Evolution of sarcomeric myosin heavy chain genes: evidence from fish

Myosin heavy chain (MYH) is a major structural protein, integral to the function of sarcomeric muscles. We investigated both exon-intron organization and amino acid sequence of sarcomeric MYH genes to infer their evolutionary history in vertebrates. Our results were consistent with the hypothesis that a multigene family encoded MYH proteins in the ancestral chordate, one gene ancestral to human MYH16 and its homologues and another ancestral to all other vertebrate sarcomeric MYH genes. We identified teleost homologues of mammalian skeletal and cardiac MYH genes, indicating that the ancestors of those genes were present before the divergence of actinopterygians and sarcopterygians. Indeed, the ancestral skeletal genes probably duplicated at least once before the divergence of teleosts and tetrapods. Fish homologues of mammalian skeletal MYH are expressed in skeletal tissue and homologues of mammalian cardiac genes are expressed in the heart but, unlike mammals, there is overlap between these expression domains. Our analyses inferred two other ancestral vertebrate MYH genes, giving rise to human MYH14 and MYH15 and their homologues. Relative to the skeletal and cardiac genes, MYH14 and MYH15 homologues are characterized by evolution of intron position, differences in evolutionary rate between the functionally differentiated head and rod of the myosin protein, and possible evolution of function among vertebrate classes. Tandem duplication and gene conversion appear to have played major roles in the evolution of at least cardiac and skeletal MYH genes in fish. One outcome of this high level of concerted evolution is that different fish taxa have different suites of MYH genes, i.e., true orthologs do not exist.