Sarcomeric Myosin Heavy Chain Research Articles

USP20 deletion promotes eccentric cardiac remodeling in response to pressure overload and increases mortality.

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well characterized. To define whether the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild-type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4 wk of TAC compared with WT cohorts. Both USP20-KO TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume overload states and decompensated heart failure. In contrast, WT-TAC mice displayed an increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis, and mouse mortality were augmented in USP20-KO-TAC compared with WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling, was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts, indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.NEW & NOTEWORTHY We identify ubiquitin-specific peptidase 20 (USP20) as an important enzyme that is required for cardiac homeostasis and function, particularly during myocardial pressure overload. USP20 regulates protein stability of cardiac MYH7, an essential molecular motor protein expressed in sarcomeres; loss-of-function mutations of MYH7 are associated with human hypertrophic cardiomyopathy, cardiac failure, and sudden death. Enhancing USP20 activity could be a potential therapeutic approach to prevent the development of maladaptive state of eccentric hypertrophy and heart failure.

Developmental, Physiological and Phylogenetic Perspectives on the Expression and Regulation of Myosin Heavy Chains in Craniofacial Muscles.

This review deals with the developmental origins of extraocular, jaw and laryngeal muscles, the expression, regulation and functional significance of sarcomeric myosin heavy chains (MyHCs) that they express and changes in MyHC expression during phylogeny. Myogenic progenitors from the mesoderm in the prechordal plate and branchial arches specify craniofacial muscle allotypes with different repertoires for MyHC expression. To cope with very complex eye movements, extraocular muscles (EOMs) express 11 MyHCs, ranging from the superfast extraocular MyHC to the slowest, non-muscle MyHC IIB (nmMyH IIB). They have distinct global and orbital layers, singly- and multiply-innervated fibres, longitudinal MyHC variations, and palisade endings that mediate axon reflexes. Jaw-closing muscles express the high-force masticatory MyHC and cardiac or limb MyHCs depending on the appropriateness for the acquisition and mastication of food. Laryngeal muscles express extraocular and limb muscle MyHCs but shift toward expressing slower MyHCs in large animals. During postnatal development, MyHC expression of craniofacial muscles is subject to neural and hormonal modulation. The primary and secondary myotubes of developing EOMs are postulated to induce, via different retrogradely transported neurotrophins, the rich diversity of neural impulse patterns that regulate the specific MyHCs that they express. Thyroid hormone shifts MyHC 2A toward 2B in jaw muscles, laryngeal muscles and possibly extraocular muscles. This review highlights the fact that the pattern of myosin expression in mammalian craniofacial muscles is principally influenced by the complex interplay of cell lineages, neural impulse patterns, thyroid and other hormones, functional demands and body mass. In these respects, craniofacial muscles are similar to limb muscles, but they differ radically in the types of cell lineage and the nature of their functional demands.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Distinct effects of two hearing loss–associated mutations in the sarcomeric myosin MYH7b

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere.

Regulation of the Expression of the Myosin Heavy Chain (MYH) Gene myh14 in Zebrafish Development.

The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.

Robust Regeneration of Adult Electric Pulse Fish and the Formation of Its Electric Organ from Fast Muscle Fibers

In most weakly electric fish species, the cells that make up the electric organ (EO) are called electrocytes (EC) and these derive from a subpopulation of striated muscle fibers that undergo an extreme phenotypic conversion to replace their contractile function for an electrogenic one that fish use to navigate, communicate, mate, and detect prey/predators. Our previous findings using the knifefish Sternopygus macrurus revealed that ECs form from the fusion of fast muscle fiber types during adult tail regeneration. Whether or not this EC formation process is conserved in other highly regenerative electric fish species is the focus of this study. Specifically, we used immunolabeling to begin characterizing the morphology and muscle-protein expression of mature electrocytes in the species Brachyhypopomus pinnicaudatus and the muscle fiber type composition in the distal-most tail surrounding ECs prior to tail amputation. Regeneration of skeletal muscle and EO tissues was studied at different time points post tail cut – 1, 2, 3, 4, and 6 weeks. Our analyses to date show a distinct sexual dimorphism in EC length and cross-sectional area (CSA), with a mean (±SE) CSA of 4718µm2±543 in males (n= 3; triplicates per fish) and 1763µm2±181 in females (n=3; triplicates per fish) and a mean EC length of 19µm±2 in males (n= 3; triplicates per fish) and 13µm±1 in females (n= 3; triplicates per fish). Our immunolabeling studies detected the presence of the “muscle” protein desmin in all adult ECs. In addition, some but not all, ECs contained alpha-actinin, tropomyosin, titin, and sarcomeric myosin heavy chain (MHC) IIb. In B. pinnicaudatus, cell differentiation progressed from proximal to distal and peripheral to central, with muscle fibers being detected prior to ECs at each stage of regeneration studied. Myogenesis was evident by Day 7 with presence of fibers positively labeled with mouse antibody (MF20) against all MHC isoforms (n= 2). In addition, by Day 7, regenerating ECs were also evident in 1-week blastemas (n=2) fish based on the clustering of myofibers stained by cresyl violet and these early ECs were immunolabeled with MF20, cell shape transformation, and medial location relative to the muscle fibers. These data suggest that there are sexual dimorphic differences in the CSAs and lengths of ECs in the EOs of male and female B. pinnicaudatus. In addition, similar to S. macrurus, developing ECs in B. pinnicaudatus are given rise to by their type II fast muscle fiber precursors.

Maintenance of the Undifferentiated State in Myogenic Progenitor Cells by TGFβ Signaling is Smad Independent and Requires MEK Activation.

Transforming growth factor β (TGFβ) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFβ potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFβ canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFβ is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFβ did not reverse TGFβ’s inhibitory effect on myogenesis. In considering other pathways, we observed that TGFβ potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFβ’s inhibitory effect on myogenesis, as indicated by a myogenin promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFβ activation. Taken together, these observations support a model in which TGFβ activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.

Quantitative Phenotyping of Xenopus Embryonic Heart Pathophysiology Using Hemoglobin Contrast Subtraction Angiography to Screen Human Cardiomyopathies.

Congenital heart disease (CHD) is a significant cause of mortality in infants and adults. Currently human genomic analysis has identified a number of candidate genes in these patients. These genes span diverse categories of gene function suggesting that despite the similarity in cardiac lesion, the underlying pathophysiology may be different. In fact, patients with similar CHDs can have drastically different outcomes, including a dramatic decrease in myocardial function. To test these human candidate genes for their impact on myocardial function, we need efficient animals models of disease. For this purpose, we paired Xenopus tropicalis with our microangiography technique, hemoglobin contrast subtraction angiography (HCSA). To demonstrate the gene-teratogen-physiology relationship, we modeled human cardiomyopathy in tadpoles. First we depleted the sarcomeric protein myosin heavy chain 6 (myh6) expression using morpholino oligos. Next, we exposed developing embryos to the teratogen ethanol and in both conditions showed varying degrees of cardiac dysfunction. Our results demonstrate that HCSA can distinguish biomechanical phenotypes in the context of gene dysfunction or teratogen. This approach can be used to screen numerous candidate CHD genes or suspected teratogens for their effect on cardiac function.

Metformin Enhances Autophagy and Provides Cardioprotection in δ-Sarcoglycan Deficiency-Induced Dilated Cardiomyopathy.

Metformin is a popular antidiabetic agent that is also used to treat heart failure patients with type 2 diabetes mellitus. Several reports suggest that metformin may also have cardioprotective effects in patients without diabetes mellitus. In the present study, we investigated the possible therapeutic effect of metformin in heart failure and its underlying molecular mechanisms using a δ-sarcoglycan-deficient mouse model of dilated cardiomyopathy. Thirty-two-week-old δ-sarcoglycan-deficient mice exhibiting established cardiomyopathy with extensive left ventricular dilatation and dysfunction were administered saline or metformin (200 mg/kg per day) for 4 weeks using osmotic mini-pumps. Metformin partially reversed the left ventricular dilatation (reverse remodeling) and significantly improved cardiac function. The hearts of metformin-treated mice showed less fibrosis, less cardiomyocyte hypertrophy, and fewer degenerative subcellular changes than saline-treated mice. These effects were accompanied by restored expression of the sarcomeric proteins myosin heavy chain and troponin I, and their transcription factor, GATA-4. Autophagy was enhanced in the hearts from metformin-treated mice, as indicated by increase of myocardial microtubule-associated protein-1 LC-3 (light chain 3)-II levels and LC3-II/-I ratios as well as levels of cathepsin D and ATP. In addition, increased numbers of autophagic vacuoles and lysosomes were accompanied increased AMP-activated protein kinase activity and suppression of mammalian target of rapamycin phosphorylation. Finally, autophagic flux assays using short-term chloroquine treatment revealed that autophagy was activated in δ-sarcoglycan-deficient hearts and was further augmented by metformin treatment. Metformin is a beneficial pharmacological tool that mitigates heart failure caused by δ-sarcoglycan deficiency in association with enhanced autophagy.

Abstract 212: Adrenergic Signaling Genes Are Not All Expressed During Myogenesis in Individual Human iPSC-derived Cardiomyocytes

Background: Adrenergic receptors (AR) in an individual cardiomyocyte (CM) are not uniformly expressed at the single-cell level (Myagmar et al., 2017). The timing and cellular distribution of AR signaling (ARS) genes in individual human iPSC-derived CMs (hiPSC-CMs) have not yet been reported. Objective: To map out the transcription program of ARS genes during hiPSC-CM myogenesis using single-cell transcriptomics (scRNA Seq.). Methods: 132 ARS in CM genes curated by the Kyoto Encyclopedia of Genes and Genomes were studied. The CMs were derived from 2 commercially available hiPSC lines using sequential GSK3 and Wnt signaling inhibition. The CMs were assayed by flow cytometry with sarcomeric myosin heavy chain (MYHC6/7) protein, a biomarker of myogenesis. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. Data from 10 somatic cell preparations and iPSCs defined the signal specificity and biological noise in the system. The expression patterns were categorized as induced, repressed, or neither based on the median transcript per million for individual cells normalized to iPSCs. A p -value of <0.05 by ANOVA was used for significance. Results: The CMs had spontaneous contractions by D14 and increased from <1% in iPSCs to 21%, 37% and 59% at D14, D30 and D60 post differentiation (adj. p <0.03) respectively. None of the iPSCs had MYH6/7 transcripts, while >90% of differentiating cells did. MYH6/7 median expression increased 15-fold from D14 to D30 (p<0.0001) and 60-fold to D60 (p<0.06). Of 132 ARS genes, 60 (44%) had no detectable expression out to D60. Of the 72 genes with expression, 18 (25%) were induced in parallel to MYH6/7, while 22 (31%) were repressed. The remaining 32 genes (44%) had no distinctive pattern. From 18 induced genes (14% of all), 8 were sarcomeric genes that preceded expression to most ARS genes without induction (86% of all). Conclusion: Single-cell transcriptomes in derived hiPSC-CMs revealed a lack of uniformity between upstream ARS genes and early sarcomeric genes. Defining the timing and cell-to-cell variability of the ARS gene program will be critical to understanding its mechanistic relationship to the sarcomeric gene program in humans.

Abstract 213: Sarcolemma Genes Related to the Transverse Tubule Structure Are Mostly Not Expressed in Differentiating Human iPSC-derived Cardiomyocytes

Background: Transverse tubules (TT) are tunnel-like extensions of sarcolemma studded with ion channels coupling excitation, through the cytoplasm, to contraction in the sarcomere of matured cardiomyocytes (CMs). Expression timing of sub-cellular TT-related genes (TT-rgs) in individual human iPSC-derived CMs (hiPSC-CMs) has not yet been reported. Objective: Map out the gene program of TT-rgs by sub-cellular locations during hiPSC-CM differentiation using single-cell transcriptomics (scRNA Seq.). Methods: hiPSC-CMs were differentiated from 2 commercially available lines using sequential GSK3 and Wnt signaling inhibition. The %CMs were assayed by flow cytometry for sarcomeric myosin heavy chain protein, a CM biomarker. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. We categorized 73 TT-rgs by cellular location of the coded protein (i.e. sarcomere, cytoplasm or sarcolemma). The expression pattern for each location was categorized as induced, repressed, or neither based on the median transcript per million for each individual cell normalized to iPSCs. Results: CMs increased from <1% in iPSCs to 21%, 37% and 59% at D14, D30 and D60 post differentiation (adj. p <0.03 by ANOVA), respectively. 33 genes (45%) had no detectable RNA while 21 (29%) were induced to D60. See table. Conclusion: Single-cell transcriptomes in differentiating hiPSC-CMs revealed a discordance between sarcolemma (mostly not expressed) and sarcomeric genes being induced. Defining timing and within cell variability of TT sub-cellular genes will be critical to understand human CM maturation.

Knockdown of subunit 3 of the COP9 signalosome inhibits C2C12 myoblast differentiation via NF-KappaB signaling pathway

BackgroundThe COP9 signalosome (CSN) is a conserved protein complex composed of 8 subunits designated CSN1-CSN8. CSN3 represents the third subunit of the CSN and maintains the integrity of the complex. CSN3 binds to the striated muscle-specific β1D integrin tail, and its subcellular localization is altered in differentiated skeletal muscle cells. However, the role of CSN3 in skeletal muscle differentiation is unknown. The main goal of this study was to identify whether CSN3 participates in myoblast differentiation and the signalling mechanisms involved using C2C12 cells as a skeletal muscle cell model.MethodsSmall-hairpin (shRNA) was used to knockdown CSN3 in C2C12 cells. Differentiation was evaluated by immunostaining and confocal microscopy. Markers of differentiation, NF-κB signaling and CSN subunits expression, were assessed by immunoblotting and/or immunostaining. Cell proliferation was analysed by cell counting, flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by one or two-way analysis of variance (ANOVA) followed by post-hoc testing.ResultsTransduction of C2C12 cells with two distinct CSN3 shRNAs led to the production of two cells lines expressing 7% of CSN3 protein (shCSN3-Low) and 43% of CSN3 protein (CSN3-Med) compared to controls. Knockdown of CSN3 was accompanied by destabilization of several CSN subunits and increased nuclear NF-κB localization. shCSN3-Med cells expressed less myogenin and formed shorter and thinner myotubes. In contrast, the shCSN3-Low cells expressed higher levels of myogenin prior and during the differentiation and remained mononucleated throughout the differentiation period. Both CSN3 knockdown cell lines failed to express sarcomeric myosin heavy chain (MHC) protein during differentiation. The fusion index was significantly higher in control cells than in shCSN3-Med cells, whereas shCSN3-Low cells showed no cell fusion. Interestingly, CSN3 knockdown cells exhibited a significantly slower growth rate relative to the control cells. Cell cycle analysis revealed that CSN3 knockdowns delayed in S phase and had increased levels of nuclear p21/Cip1 and p27/Kip1.ConclusionsThis study clarifies the first step toward unrevealing the CSN3/CSN-mediated pathways that controls C2C12 differentiation and proliferation. Further in vivo characterization of CSN/CSN3 may lead to the discovery of novel therapeutic target of skeletal muscle diseases such as muscular dystrophies.

Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species

In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species.To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b).

Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)—a co-activator not only for serum response factor, but also for Gata4 and Tbx5—is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

Evolution of the myosin heavy chain gene MYH14 and its intronic microRNA miR-499: muscle-specific miR-499 expression persists in the absence of the ancestral host gene

BackgroundA novel sarcomeric myosin heavy chain gene, MYH14, was identified following the completion of the human genome project. MYH14 contains an intronic microRNA, miR-499, which is expressed in a slow/cardiac muscle specific manner along with its host gene; it plays a key role in muscle fiber-type specification in mammals. Interestingly, teleost fish genomes contain multiple MYH14 and miR-499 paralogs. However, the evolutionary history of MYH14 and miR-499 has not been studied in detail. In the present study, we identified MYH14/miR-499 loci on various teleost fish genomes and examined their evolutionary history by sequence and expression analyses.ResultsSynteny and phylogenetic analyses depict the evolutionary history of MYH14/miR-499 loci where teleost specific duplication and several subsequent rounds of species-specific gene loss events took place. Interestingly, miR-499 was not located in the MYH14 introns of certain teleost fish. An MYH14 paralog, lacking miR-499, exhibited an accelerated rate of evolution compared with those containing miR-499, suggesting a putative functional relationship between MYH14 and miR-499. In medaka, Oryzias latipes, miR-499 is present where MYH14 is completely absent in the genome. Furthermore, by using in situ hybridization and small RNA sequencing, miR-499 was expressed in the notochord at the medaka embryonic stage and slow/cardiac muscle at the larval and adult stages. Comparing the flanking sequences of MYH14/miR-499 loci between torafugu Takifugu rubripes, zebrafish Danio rerio, and medaka revealed some highly conserved regions, suggesting that cis-regulatory elements have been functionally conserved in medaka miR-499 despite the loss of its host gene.ConclusionsThis study reveals the evolutionary history of the MYH14/miRNA-499 locus in teleost fish, indicating divergent distribution and expression of MYH14 and miR-499 genes in different teleost fish lineages. We also found that medaka miR-499 was even expressed in the absence of its host gene. To our knowledge, this is the first report that shows the conversion of intronic into non-intronic miRNA during the evolution of a teleost fish lineage.

2D DIGE/MS to investigate the impact of slaughtering techniques on postmortem integrity of fish filet proteins

Two-dimensional difference gel electrophoresis (2D DIGE) was applied to investigate the impact of slaughtering on the postmortem integrity of muscle tissue proteins in European sea bass (Dicentrarchus labrax). Three different slaughtering techniques were evaluated: asphyxia in air (AA), asphyxia in ice (AI), and spinal cord severance (SCS). Principal components analysis (PCA) revealed a significant divergence of SCS samples, whereas AA and AI samples, although grouped separately, were less divergent and could be included in a single asphyxia cluster. In terms of single proteins, the most significant impact was seen on nucleoside diphosphate kinase B, which was consistently less affected when fish were slaughtered by SCS as compared to asphyxia. Integrity of the sarcomeric proteins myosin heavy chain and myosin binding protein C and of the cytosolic proteins fructose biphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and enolase 1 was also better preserved upon SCS slaughtering. Most interestingly, the influence on muscle protein integrity could be detected since the early postmortem phase. In conclusion, slaughtering by SCS preserves protein integrity better than death by asphyxia, either in ice or in air. Both asphyxia conditions are comparably more adverse than SCS to muscle protein integrity, although a general trend favoring AI over AA is observed.

TGF-β1 enhances contractility in engineered skeletal muscle

Scaffoldless engineered 3D skeletal muscle tissue created from satellite cells offers the potential to replace muscle tissue that is lost due to severe trauma or disease. Transforming growth factor-beta 1 (TGF-β1) plays a vital role in mediating migration and differentiation of satellite cells during the early stages of muscle development. Additionally, TGF-β1 promotes collagen type I synthesis in the extracellular matrix (ECM) of skeletal muscle, which provides a passive elastic substrate to support myofibres and facilitate the transmission of force. To determine the role of TGF-β1 in skeletal muscle construct formation and contractile function in vitro, we created tissue-engineered 3D skeletal muscle constructs with varying levels of recombinant TGF-β1 added to the cell culture medium. Prior to the addition of TGF-β1, the primary cell population was composed of 75% Pax7-positive cells. The peak force for twitch, tetanus and spontaneous force were significantly increased in the presence of 2.0 ng/ml TGF-β1 when compared to 0, 0.5 and 1.0 ng/ml TGF-β1. Visualization of the cellular structure with H&E and with immunofluorescence staining for sarcomeric myosin heavy chains and collagen type I showed denser regions of better organized myofibres in the presence of 2.0 ng/ml TGF-β1 versus 0, 0.5 and 1.0 ng/ml. The addition of 2.0 ng/ml TGF-β1 to the culture medium of engineered 3D skeletal muscle constructs enhanced contractility and extracellular matrix organization.

Has the Genomic Revolution Failed?

Has the Genomic Revolution Failed?

Abstract 9559: The Role of Myopalladin in the Interactions of ANKRD1/CARP with the Co-Activator p53 in Hypertrophic Cardiomyopathy

Background: Myopalladin (MYPN), a member of the Z-disc that interacts with other Z-disc proteins, including the cardiac ankyrin repeat protein ANKRD1 (CARP). Mutations in MYPN and CARP genes cause human dilated (DCM) and hypertrophic cardiomyopathies (HCM). Both MYPN and CARP have dual distributions in the cytoplasm and nucleus. In the nucleus, CARP is a transcriptional co-activator of p53, repressing expression of the cardiac proteins in an in vitro system. A MYPN-dependent cytoplasmic anchoring for CARP is described although the molecular basis of the MYPN modulation of CARP gene transcription is unclear. Previously, our laboratory identified a novel MYPN variant (MYPN Y20C ) in DCM and HCM patients. A transgenic MYPN Y20C cardiac-restricted overexpression mouse model recapitulates the HCM phenotype.. Methods and Results: In vitro and in vivo studies demonstrated that the MYPN Y20C mutation resulted in perinuclear localization in myoblasts while CARP retained its cytoplasmic/nuclear dual distribution; as did wild-type MYPN. Co-immunoprecipitation revealed decreased association between CARP and MYPN Y20C suggesting a dissociation of the cellular distributions of CARP and MYPN Y20C . In transgenic MYPN Y20C hearts, perinuclear MYPN Y20C had increased association with p53, a co-activator for negative gene regulation of CARP. Nuclear localization of p53 is a critical element in the activation of its transcription. Its sequestration in the cytoplasm renders the protein non-functional. This resulted in a loss-of-function of CARP gene transcription as demonstrated by increased expression of the CARP-specific sarcomeric protein myosin heavy chain (MHC), and atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) in vivo . CARP was serine phosphorylated and coexpression with MYPN led to suppression of CARP phosphorylation. Conclusion: Our study demonstrates a novel role for myopalladin in the development of HCM. The perinuclear/cytoplasmic sequestration of p53 by MYPN Y20C resulted in the inactivation of p53, which leads to loss-of-function of CARP gene regulation. Our data also suggests that myopalladin plays a pivotal role in relaying cardiac Z-disc signaling to those proteins involved in CARP phosphorylation/activation.

Species-specific expression variation of fish MYH14, an ancient vertebrate myosin heavy chain gene orthologue

MYH14 (MYH7b) is the most recently identified sarcomeric myosin heavy chain gene (MYH) from the human genome. Recent observations have revealed that MYH M5 , a torafugu orthologue of MYH14, is one of the major components of the MYH repertoire expressed in torafugu embryos and adults, suggesting its pivotal role in fish muscle formation. We have examined the expression pattern of MYH14 in zebrafish, a model organism for the study of vertebrate development. In situ hybridization studies revealed that zebrafish MYH14 was expressed in the most myotomal region containing fast muscle fibers in the embryonic stage and in superficial slow muscle fibers in the adult stage. Weak signals of MYH14 transcripts were also detected in intermediate muscle fibers located between superficial slow and inner fast muscle fibers in the adult. Reverse transcription-PCR studies showed that MYH14 was not only expressed in skeletal muscles but also in cardiac muscles. These expression patterns are in a marked contrast to that of torafugu MYH M5 , which is solely expressed in slow and cardiac muscle fibers in both the embryonic and adult stages. Our results imply species-specific functions of MYH14 in fish muscle formation.