Rescue of skeletal muscle α-actin–null mice by cardiac (fetal) α-actin
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ABSTRACT:
SUBMITTER: Kristen J. Nowak
PROVIDER: S-JCBD-200812132 | bioimages |
REPOSITORIES: bioimages
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Project description:Skeletal muscle alpha-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac alpha-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.
| S-EPMC2711600 | biostudies-literature
Project description:Skeletal muscle actin mice (Crawford et al., (2002) Mol Cell Biol 22, 5587) were crossed with cardiac actin transgenic mice (termed "ACTC^Coco" or "Coco" for short), to produce mice that had cardiac actin instead of skeletal muscle actin in their skeletal muscles (termed "ACTC^Co/KO" or for short "Coco/KO"). Microarray analysis using the Illumina mouse-6 v1.1 expression beadchip was performed on RNA extraced from the soleus muscle of Coco/KO mice and wildtype mice, to confirm the swith in actin isoform expression, and to determine what other differences might exist between wildtype mice and the Coco/KO mice. Keywords: genetic modification 3 RNA samples (each being the pool of two individual samples extracted from different soleus muscles from different individual mice) per genotype (either wildtype or Coco/KO) were used. The total 6 RNA samples were processed using an Illumina mouse-6 v1.1expression beadchip and then the differentially expressed genes determined.
2009-05-24 | E-GEOD-12882 | biostudies-arrayexpress
Project description:Skeletal muscle actin mice (Crawford et al., (2002) Mol Cell Biol 22, 5587) were crossed with cardiac actin transgenic mice (termed "ACTC^Coco" or "Coco" for short), to produce mice that had cardiac actin instead of skeletal muscle actin in their skeletal muscles (termed "ACTC^Co/KO" or for short "Coco/KO"). Microarray analysis using the Illumina mouse-6 v1.1 expression beadchip was performed on RNA extraced from the soleus muscle of Coco/KO mice and wildtype mice, to confirm the swith in actin isoform expression, and to determine what other differences might exist between wildtype mice and the Coco/KO mice. Keywords: genetic modification
2009-05-25 | GSE12882 | GEO
Project description:Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo.
| S-EPMC4755912 | biostudies-literature
Project description:α-smooth muscle actin, encoded by ACTA2 gene, is an isoform of the vascular smooth muscle actins, typically expressed in the vascular smooth muscle cells contributing to vascular motility and contraction. ACTA2 gene mutations cause a diversity of diffuse vasculopathies such as thoracic aortic aneurysms and dissections as well as occlusive vascular diseases, including premature coronary artery disease and ischemic stroke. Dynamics of differentiation-specific α-smooth muscle actin in arterial smooth muscle cells and proliferation of the proteins have been well described. Although a variety of research works have been undertaken in terms of modifications of α-smooth muscle actin and mutations of ACTA2 gene and myosin, the underlying mechanisms towards the pathological processes by way of gene mutations are yet to be clarified. The purpose of the present article is to describe the phenotypes of α-smooth muscle actin and implications of ACTA2 mutations in vasculopathies in order to enhance the understanding of potential mechanisms of aortic and coronary disorders.
| S-EPMC4762557 | biostudies-literature
Project description:The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln(137) in these activities, we characterized Gln(137) mutants of human cardiac muscle alpha-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the gamma-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln(137) plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.
| S-EPMC3258932 | biostudies-literature
Project description:BackgroundA critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression.MethodsThe association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor β1 (TGF-β1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing.ResultsThe human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-β1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-β1 or 5'-aza.ConclusionOur data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.
| S-EPMC7263943 | biostudies-literature
Project description:The ACTA1 gene encodes skeletal muscle alpha-actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease-causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease-causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core-like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype-phenotype correlations, we have developed a locus-specific database for ACTA1 variations (http://waimr.uwa.edu.au).
| S-EPMC2784950 | biostudies-literature
Project description:Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.
| S-EPMC5862523 | biostudies-literature
Project description:Twenty-two missense mutations in ACTA2, which encodes ?-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human ?-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.
| S-EPMC3064192 | biostudies-literature