Muscle Myosin Heavy Chain Gene Research Articles

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

Effects of Atmospheric Ammonia on Skeletal Muscle Growth in Broilers

Ammonia, one of the most polluted gases in poultry houses, has always been an urgent problem to solve. Exposure to ammonia can threaten the respiratory tract, induce inflammation, and decrease growth performance. To date, there are few studies investigating the effects of ammonia on skeletal muscle growth. In this experiment, a total of 144 broilers were randomly divided into two groups, and 0 ppm and 35 ppm atmospheric ammonia were administered in the chambers. The trial lasted for 21 days. The breast muscle, thigh muscle, dressed weight, and serum biochemical indexes were measured. The skeletal muscle fibre morphology was observed using light microscopy, and the expressions of genes associated with skeletal muscle development and myosin heavy chain genes were assessed. After 7 days of ammonia exposure, the broilers’ weight in the ammonia group decreased. On the 21st day of the experiment, in the ammonia group, the breast muscle weight, thigh muscle weight, and dressed weight decreased, the blood urea nitrogen content increased, skeletal muscle fibre diameter shortened, the expression of myostatin increased, and the expression of myosin heavy chain-FWM and myosin heavy chain-FRM decreased significantly. This article suggests that 35 ppm atmospheric ammonia seriously affects the skeletal muscle gain rate of broilers, and the myostatin pathway could be a potential regulation of the growth rate of muscle fibre under ammonia exposure.

MEF2B is the ideal immunohistochemical marker to highlight neoplastic LP cells in nodular lymphocyte-predominant Hodgkin lymphoma.

MEF2B is the ideal immunohistochemical marker to highlight neoplastic LP cells in nodular lymphocyte-predominant Hodgkin lymphoma.

Histopathological, Ultrastructural, and Immunohistochemical Findings in MYH11-Variant Visceral Myopathy.

Pathogenic mutations in the smooth muscle myosin heavy chain gene, MYH11, cause megacystis megacolon intestinal hypoperistalsis syndrome and other forms of chronic intestinal pseudo-obstruction. Evaluation of intestinal tissues from affected patients is often performed before mutational analysis, but the pathological findings of MYH11-variant visceral myopathy have not been well defined. Light microscopic, immunohistochemical, and ultrastructural findings from multiple intestinal samples from 2 patients with MYH11-variant visceral myopathy were reviewed, including MYH11-specific immunohistochemistry. The findings were compared with intestinal samples from patients with gamma-smooth muscle actin (ACTG2)-variant visceral myopathy and non-pseudo-obstruction controls. Apart from non-specific changes (e.g., muscle hypertrophy and distension-related muscularis propria necrosis), no alterations were identified by routine histopathological evaluation or electron microscopy. Immunohistochemistry with antibodies against a battery of smooth muscle proteins, including MYH11, revealed indistinguishable patterns of immunoreactivity in the muscularis propria of both patients and controls. Myopathic morphological or immunohistochemical changes may not be present in intestinal specimens from patients with MYH11-variant visceral myopathy. Molecular genetic studies should be considered for patients with chronic intestinal pseudo-obstruction and normal or non-specific pathology findings.

The effects of progenitor and differentiated cells on ectopic calcification of engineered vascular tissues

Ectopic vascular calcification associated with aging, diabetes mellitus, atherosclerosis, and chronic kidney disease is a considerable risk factor for cardiovascular events and death. Although vascular smooth muscle cells are primarily implicated in calcification, the role of progenitor cells is less known. In this study, we engineered tubular vascular tissues from embryonic multipotent mesenchymal progenitor cells either without differentiating or after differentiating them into smooth muscle cells and studied ectopic calcification through targeted gene analysis. Tissues derived from both differentiated and undifferentiated cells calcified in response to hyperphosphatemic inorganic phosphate (Pi) treatment suggesting that a single cell-type (progenitor cells or differentiated cells) may not be the sole cause of the process. We also demonstrated that Vitamin K, which is the matrix gla protein activator, had a protective role against calcification in engineered vascular tissues. Addition of partially-soluble elastin upregulated osteogenic marker genes suggesting a calcification process. Furthermore, partially-soluble elastin downregulated smooth muscle myosin heavy chain (Myh11) gene which is a late-stage differentiation marker. This latter point, in turn, suggests that SMC may be switching into a synthetic phenotype which is one feature of vascular calcification. Taken together, our approach presents a valuable tool to study ectopic calcification and associated gene expressions relevant to clinical therapeutic targets.

Identification and classification of interstitial cells in the mouse renal pelvis.

Platelet-derived growth factor receptor-α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed 'atypical' smooth muscle cells) in the murine and cynomolgus monkey pelvis-kidney junction. PDGFRα+ cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres. Most c-Kit+ cells in the renal pelvis are mast cells. Mast cells (CD117+ /CD45+ ) are more abundant in the proximal renal pelvis and pelvis-kidney junction regions whereas c-Kit+ interstitial cells (CD117+ /CD45- ) are found predominantly in the distal renal pelvis and ureteropelvic junction. PDGFRα+ cells are distinct from c-Kit+ interstitial cells. A subset of PDGFRα+ cells express the Ca2+ -activated Cl- channel, anoctamin-1, across the entire renal pelvis. Spontaneous Ca2+ transients were observed in c-Kit+ interstitial cells, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using mice expressing genetically encoded Ca2+ sensors. Rhythmic contractions of the renal pelvis transport urine from the kidneys into the ureter. Specialized pacemaker cells, termed atypical smooth muscle cells (ASMCs), are thought to drive the peristaltic contractions of typical smooth muscle cells (TSMCs) in the renal pelvis. Interstitial cells (ICs) in close proximity to ASMCs and TSMCs have been described, but the role of these cells is poorly understood. The presence and distributions of platelet-derived growth factor receptor-α+ (PDGFRα+ ) ICs in the pelvis-kidney junction (PKJ) and distal renal pelvis were evaluated. We found PDGFRα+ ICs in the adventitial layers of the pelvis, the muscle layer of the PKJ and the adventitia of the distal pelvis. PDGFRα+ ICs were distinct from c-Kit+ ICs in the renal pelvis. c-Kit+ ICs are a minor population of ICs in murine renal pelvis. The majority of c-Kit+ cells were mast cells. PDGFRα+ cells in the PKJ co-expressed smooth muscle myosin heavy chain (smMHC) and several other smooth muscle gene transcripts, indicating these cells are ASMCs, and PDGFRα is a novel biomarker for ASMCs. PDGFRα+ cells also express Ano1, which encodes a Ca2+ -activated Cl- conductance that serves as a primary pacemaker conductance in ICs of the GI tract. Spontaneous Ca2+ transients were observed in c-Kit+ ICs, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using genetically encoded Ca2+ sensors. A reporter strain of mice with enhanced green fluorescent protein driven by the endogenous promotor for Pdgfra was shown to be a powerful new tool for isolating and characterizing the phenotype and functions of these cells in the renal pelvis.

HDAC1 Is a Required Cofactor of CBFβ-SMMHC and a Potential Therapeutic Target in Inversion 16 Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a neoplastic disease characterized by the uncontrolled proliferation and accumulation of immature myeloid cells. A common mutation in AML is the inversion of chromosome 16 [inv (16)], which generates a fusion between the genes for core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11), forming the oncogene CBFB-MYH11. The expressed protein, CBFβ-SMMHC, forms a heterodimer with the key hematopoietic transcription factor RUNX1. Although CBFβ-SMMHC was previously thought to dominantly repress RUNX1, recent work suggests that CBFβ-SMMHC functions together with RUNX1 to activate transcription of specific target genes. However, the mechanism of this activity or a requirement for additional cofactors is not known. Here, we show that the epigenetic regulator histone deacetylase 1 (HDAC1) forms a complex with CBFβ-SMMHC, colocalizes with RUNX1 and CBFβ-SMMHC on the promoters of known fusion protein target genes, and that Hdac1 is required for expression of these genes. These results imply that HDAC1 is an important component of the CBFβ-SMMHC transcriptional complex, and that leukemia cells expressing the fusion protein may be sensitive to treatment with HDAC1 inhibitors. Using a knock-in mouse model expressing CBFβ-SMMHC, we found that in vivo treatment with the HDAC1 inhibitor entinostat decreased leukemic burden, and induced differentiation and apoptosis of leukemia cells. Together, these results demonstrate that HDAC1 is an important cofactor of CBFβ-SMMHC and a potential therapeutic target in inv (16) AML. IMPLICATIONS: This report describes a novel role for HDAC1 as a cofactor for the leukemogenic fusion protein CBFβ-SMMHC and shows that inhibitors of HDAC1 effectively target leukemia cells expressing the fusion protein in vivo.

Improved bladder smooth muscle cell differentiation of the mesenchymal stem cells when grown on electrospun polyacrylonitrile/polyethylene oxide nanofibrous scaffold.

Reconstruction of the bladder wall plays an important role in improving its function in patients with urinary bladder dysfunction. Tissue engineering has been trying to introduce biocompatible nanofibers as scaffolds for bladder wall matrix substitutes. In this study a composite nanofibrous scaffold was fabricated from polyacrylonitrile (PAN) and polyethylene oxide (PEO) blend by electrospinning method and then its morphological and mechanical characteristics was evaluated by scanning electron microscopy (SEM), tensile, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Then smooth muscle cell (SMC) differentiation supportive capacity of PAN-PEO nanofibers was investigated by culturing of human adipose tissue-derived mesenchymal stem cells (AT-MSCs) on this scaffold and then its differentiation potential in different groups was investigated using SMC-related gene and protein markers. SEM and MTT results demonstrated that PAN-PEO supported AT-MSCs attachment, growth and proliferation, especially at early times after cell seeding. The obtained results from real-time reverse transcription polymerase chain reaction revealed that collagen-I-α1, collagen-III-α1, α-smooth muscle actin (α-SMA), calponin1, SM22α, caldesmon1, elastin, and myosin heavy chain (MHC) genes were expressed in AT-MSCs cultured on PAN-PEO significantly higher than those stem cells that cultured on the culture plate as a control. In addition α-SMA and MHC proteins were also expressed in AT-MSCs cultured on PAN-PEO significantly higher than control. According to the results PAN-PEO nanofibrous scaffold showed a positive AT-MSCs-seeded PAN-PEO has a great promising potential to use in bladder tissue engineering applications.

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

Serotonin augments smooth muscle differentiation of bone marrow stromal cells

Bone marrow stromal cells (BMSCs) contain a subset of multipotent stem cells. Here, we demonstrate that serotonin, a biogenic amine released by platelets and mast cells, can induce the smooth muscle differentiation of BMSCs. Brown Norway rat BMSCs stimulated with serotonin had increased expression of the smooth muscle markers smooth muscle myosin heavy chain (MHC) and α actin (α-SMA) by qPCR and Western blot, indicating smooth muscle differentiation. This was accompanied by a concomitant down-regulation of the microRNA miR-25-5p, which was found to negatively regulate smooth muscle differentiation. Serotonin upregulated serum response factor (SRF) and myocardin, transcription factors known to induce contractile protein expression in smooth muscle cells, while it down-regulated Elk1 and Kruppel-like factor 4 (KLF4), known to induce proliferation. Serotonin increased SRF binding to promoter regions of the MHC and α-SMA genes, assessed by chromatin immunoprecipitation assay. Induction of smooth muscle differentiation by serotonin was blocked by the knock-down of SRF and myocardin. Transforming growth factor (TGF)-β1 was constitutively expressed by BMSCs and serotonin triggered its release. Inhibition of miR-25-5p augmented TGF-β1 expression, however the differentiation of BMSCs was not mediated by TGF-β1. These findings demonstrate that serotonin promotes a smooth muscle-like phenotype in BMSCs by altering the balance of SRF, myocardin, Elk1 and KLF4 and miR-25-5p is involved in modulating this balance. Therefore, serotonin potentially contributes to the pathogenesis of diseases characterized by tissue remodeling with increased smooth muscle mass.

Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times within bilaterian evolution.

Abstract 3850: The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis.

Abstract Chromosome 16 inversion is associated with acute myeloid leukemia subtype M4Eo and produces a fusion gene CBFB-MYH11 that contains part of the core binding factor (CBF) β gene CBFB, and part of the smooth muscle myosin heavy chain (SMMHC) gene MYH11. This fusion gene encodes a fusion protein CBFβ-SMMHC, which is oncogenic and binds to the runt domain (RD) of RUNX1, another member of the CBF transcription factor family, resulting in repression of RUNX1 transactivation. We have generated mouse models by conventional and conditional knock-in of the Cbfb-MYH11 fusion gene and demonstrated that Cbfb-MYH11 represses Runx1 function in hematopoiesis and predisposes mice to myeloid leukemia (Castilla et. al., Cell 1996; Nat Genet, 1999). RUNX1 binding and repression was previously considered a key step in leukemogenesis by CBFβ-SMMHC. In order to dissect the molecular mechanism of RUNX1 and CBFβ-SMMHC interaction during leukemogenesis, we generated a knock-in mouse model with deleted high affinity binding site of Cbfb-MYH11. We found accelerated leukemia development in these mice (Kamikubo et.al., Cancer cell, 2010) suggesting that Cbfb-MYH11 play an independent role apart from Runx1 binding and repression. To test if Runx1 is involved in the leukemia development and progression, we crossed Cbfb-MYH11 knock-in mice with mice harboring one of the two mutant alleles of Runx1 - Runx1+/- and Runx1+/Lzd. Runx1+/- contains a null allele while Runx1+/Lzd contains a knocked-in fusion between the RD of Runx1 and the LacZ gene, which is partially dominant-negative in reporter assays. We have determined the rate and percentage of leukemia development in these mice. We found that the Cbfb-MYH11 mice that were Runx1+/- had a similar rate of leukemogenesis when compared with Cbfb-MYH11 mice that were Runx1+/+. However, the Cbfb-MYH11 mice that were Runx1+/Lzd had significantly delayed leukemogenesis as compared to Cbfb-MYH11 mice that were Runx1+/+. Moreover, some of the Cbfb-MYH11; Runx1+/Lzd mice did not develop leukemia at the end of the one-year observation. We detected a decrease of BrdU incorporation in the bone marrow cells in mice with the Runx1+/Lzd allele, suggesting that the delayed leukemia development resulted, at least in part, from decreased proliferation. These data demonstrated that Runx1 is likely required for leukemogenesis by CBFβ-SMMHC. Citation Format: Ling Zhao, R Katherine Hyde, Lemlem Alemu, P Paul Liu. The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2013-3850

An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice

During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues

Myotonic dystrophy type 1 (DM1) is a complex multisystemic disorder caused by an expansion of a CTG repeat located at the 3′ untranslated region (UTR) of DMPK on chromosome 19q13.3. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms of DM1 including insulin resistance, muscle wasting and myotonia. In this paper we analyzed the expression of the MYH14 mRNA and protein in the muscle of DM1 patients (n=12) with different expansion lengths and normal subjects (n=7). The MYH14 gene is located on chromosome 19q13.3 and encodes for one of the heavy chains of the so called class II “nonmuscle” myosins (NMHCII). MYH14 has two alternative spliced isoforms: the inserted isoform (NMHCII-C1) which includes 8 amino acids located in the globular head of the protein, not encoded by the non inserted isoform (NMHCII-C0). Results showed a splicing unbalance of the MYH14 gene in DM1 muscle, with a prevalent expression of the NMHCII-C0 isoform more marked in DM1 patients harboring large CTG expansions. Minigene assay indicated that levels of the MBNL1 protein positively regulates the inclusion of the MYH14 exon 6. Quantitative analysis of the MYH14 expression revealed a significant reduction in the DM1 muscle samples, both at mRNA and protein level. No differences were found between DM1 and controls in the skeletal muscle localization of MYH14, obtained through immunofluorescence analysis. In line with the thesis of an “RNA gain of function” hypothesis described for the CTG mutation, we conclude that the alterations of the MYH14 gene may contribute to the DM1 molecular pathogenesis.

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Novel epigenetic regulation of skeletal muscle myosin heavy chain genes. Focus on “Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading”

skeletal muscle is a highly plastic tissue, capable of responding and adapting to a variety of physiological stimuli, e.g., muscle loading and unloading. Multiple functional phenotypes of skeletal muscle exist throughout the body at the same time. For example, muscles required for antigravity support of the skeleton or sustained locomotion are generally slow-contracting, low force-generating, and relatively resistant to fatigue, while other muscles required for quick, powerful movements are generally fast-contracting, high force-generating, and easily fatigued. In fact, skeletal muscle phenotypes have been characterized using combinations of muscle properties, including metabolic, fatigability, color, innervation, or predominant contractile protein expression. Myosin heavy chain (MHC) is the most abundant protein in skeletal muscle, comprising ∼25% of the total protein content, and is a major component of the complex responsible for generating contractile force in skeletal muscle. At least nine MHC isoforms, each transcribed from their own gene, exist in mammalian striated muscle; but only slow type I MHC and various isoforms of fast type II (IIa, IIx/d, and IIb MHC isoforms) are present in adult limb skeletal muscle that is not undergoing regeneration (16). It is well established that skeletal muscle phenotype, particularly MHC gene expression, can be altered by different states of muscular activity or inactivity, as well as various hormonal and metabolic factors (1).

Type B Core Binding Factor β/Smooth Muscle Myosin Heavy Chain Fusion Transcript in Myeloid Blast Phase of Chronic Myeloid Leukemia: Correlation with Nuclear and Cytoplasmic Localization of the Fusion Protein

BackgroundInv(16)(p13q22) or the rare t(16;16)(p13;q22) is characteristic of the M4Eo type of acute myeloid leukemia and results in the fusion of the core binding factor β gene (CBFB) at 16q22 with the smooth muscle myosin heavy chain gene (MYH11) at 16p13. The fusion transcript CBFB/MYH11 plays an important role in leukemogenesis. Many variations of this fusion transcript have been reported, with type A being most common and occurring in > 85% of cases. Patients and MethodsWe studied the fusion transcripts in 4 cases of chronic myelogenous leukemia in myeloid blast phase with inv(16)(p13q22). ResultsSequencing analysis revealed type B fusion transcript in 3 of the 4 cases. Immunohistochemistry demonstrated both nuclear and cytoplasmic staining for the fusion protein. ConclusionAlthough the biologic significance of this finding is unknown, the high frequency of an unusual type of fusion transcript in the chronic myelogenous leukemia cases in blast phase in which inv(16)(p13q22) first occurred at the time of blast transformation suggests that the type B transcript might play a role in disease progression. The effect of the additional smooth muscle myosin heavy chain sequences in the type B fusion on subcellular localization of the protein product is also suggested.