Myosin Heavy Chain Protein Research Articles

Effect of Maternal Steroid on Developing Diaphragm Integrity

Antenatal steroids reduce the severity of initial respiratory distress of premature newborn babies but may have an adverse impact on other body organs. The study aimed to examine the effect of maternal steroids on postnatal respiratory muscle function during development and elucidate the mechanisms underlying the potential myopathy in newborn rats. Pregnant rats were treated with intramuscular injections of 0.5 mg/kg betamethasone 7 d and 3 d before birth. Newborn diaphragms were dissected for assessment of contractile function at 2 d, 7 d or 21 d postnatal age (PNA), compared with age-matched controls. The expression of myosin heavy chain (MHC) isoforms and atrophy-related genes and activity of intracellular molecular signalling were measured using quantitative PCR and/or Western blot. With advancing PNA, neonatal MHC gene expression decreased progressively while MHC IIb and IIx isoforms increased. Protein metabolic signalling showed high baseline activity at 2 d PNA, and significantly declined at 7 d and 21 d. Antenatal administration of betamethasone significantly decreased diaphragm force production, fatigue resistance, total fast fibre content and anabolic signalling activity (Akt and 4E-BP1) in 21 d diaphragm. These responses were not observed in 2 d or 7 d postnatal diaphragm. Results demonstrate that maternal betamethasone treatment causes postnatal diaphragmatic dysfunction at 21 d PNA, which is attributed to MHC II protein loss and impairment of the anabolic signalling pathway. Developmental modifications in MHC fibre composition and protein signalling account for the age-specific diaphragm dysfunction.

Regenerative defect in vastus lateralis muscle of patients with chronic obstructive pulmonary disease.

BackgroundImpaired skeletal muscle regeneration could contribute to the progression of muscle atrophy in patients with chronic obstructive pulmonary disease (COPD).MethodsSatellite cells and myogenesis-related proteins were compared between healthy subjects and patients with COPD, with or without muscle atrophy. Satellite cells were isolated and cultured to assess their proliferative and differentiation aptitudes.ResultsAlthough satellite cell numbers in muscle samples were similar between groups, the proportion of muscle fibers with central nuclei was increased in COPD. In muscle homogenates, increased expression of MyoD and decreased expression of myogenin and MRF4 were observed in COPD. In cultured satellite cells of patients with COPD, increased protein content was observed for Pax7, Myf5 (proliferation phase) and myogenin (differentiation phase) while myosin heavy chain protein content was significantly lower during differentiation.ConclusionIn COPD, the number of central nuclei was increased in muscle fibers suggesting a greater number of attempts to regenerate muscle tissue than in healthy subjects. Myogenesis signaling was also altered in muscle homogenates in patients with COPD and there was a profound reduction in the differentiation potential in this population as indicated by a reduced ability to incorporate myosin heavy chain into newly formed myotubes. Collectively, these results indicate that skeletal muscle regenerative capacity termination is impaired in COPD and could contribute to the progression of muscle atrophy progression in this population.

Water-based oligochitosan and nanowhisker chitosan as potential food preservatives for shelf-life extension of minced pork

Water-based chitosans in the forms of oligochitosan (OligoCS) and nanowhisker chitosan (CSWK) are proposed as a novel food preservative based on a minced pork model study. The high surface area with a positive charge over the neutral pH range (pH 5–8) of OligoCS and CSWK lead to an inhibition against Gram-positive (Staphylococcusaureus, Listeriamonocytogenes, and Bacilluscereus) and Gram-negative microbes (Salmonellaenteritidis and Escherichiacoli O157:H7). In the minced pork model, OligoCS effectively performs a food preservative for shelf-life extension as clarified from the retardation of microbial growth, biogenic amine formation and lipid oxidation during the storage. OligoCS maintains almost all myosin heavy chain protein degradation as observed in the electrophoresis. The present work points out that water-based chitosan with its unique morphology not only significantly inhibits antimicrobial activity but also maintains the meat quality with an extension of shelf-life, and thus has the potential to be used as a food preservative.

Resveratrol Inhibits Phenotypic Switching of Neointimal Vascular Smooth Muscle Cells After Balloon Injury Through Blockade of Notch Pathway

Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action. Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days. Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls. The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway.

Dynamics of myosin degradation in intensive care unit-acquired weakness during severe critical illness

Intensive care unit (ICU)-acquired muscle wasting is a devastating complication leading to persistent weakness and functional disability. The mechanisms of this myopathy are unclear, but a disturbed balance of myosin heavy chain (MyHC) is implicated. To investigate pathways of myosin turnover in severe critically ill patients at high risk of ICU-acquired weakness. Prospective, mechanistic, observational study. Interdisciplinary ICUs of a university hospital. Twenty-nine patients with Sequential Organ Failure Assessment (SOFA) scores of at least 8 on three consecutive days within the first 5 days in ICU underwent two consecutive open skeletal muscle biopsies from the vastus lateralis at median days 5 and 15. Control biopsy specimens were from healthy subjects undergoing hip-replacement surgery. None. Time-dependent changes in myofiber architecture, MyHC synthesis, and degradation were determined and correlated with clinical data. ICU-acquired muscle wasting was characterized by early, disrupted myofiber ultrastructure followed by atrophy of slow- and fast-twitch myofibers at later time points. A rapid decrease in MyHC mRNA and protein expression occurred by day 5 and persisted at day 15 (P < 0.05). Expression of the atrophy genes MuRF-1 and Atrogin1 was increased at day 5 (P < 0.05). Early MuRF-1 protein content was closely associated with late myofiber atrophy and the severity of weakness. Decreased synthesis and increased degradation of MyHCs contribute to ICU-acquired muscle wasting. The rates and time frames suggest that pathogenesis of muscle failure is initiated very early during critical illness. The persisting reduction of MyHC suggests that sustained treatment is required.

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.

Angiotensin II and the JNK pathway mediate urotensin II expression in response to hypoxia in rat cardiomyocytes

Cardiomyocyte hypoxia causes cardiac hypertrophy through cardiac-restricted gene expression. Urotensin II (UII) cooperates with activating protein 1 (AP1) to regulate cardiomyocyte growth in response to myocardial injuries. Angiotensin II (AngII) stimulates UII expression, reactive oxygen species (ROS) production, and cardiac hypertrophy. This study aimed to evaluate the expression of UII, ROS, and AngII as well as their genetic transcription after hypoxia treatment in neonatal cardiomyocytes. Cultured neonatal rat cardiomyocytes were subjected to hypoxia for different time periods. UII (Uts2) protein levels increased after 2.5% hypoxia for 4 h with earlier expression of AngII and ROS. Both hypoxia and exogenously added AngII or Dp44mT under normoxia stimulated UII expression, whereas AngII receptor blockers, JNK inhibitors (SP600125), JNK siRNA, or N-acetyl-l-cysteine (NAC) suppressed UII expression. The gel shift assay indicated that hypoxia induced an increase in DNA-protein binding between UII and AP1. The luciferase assay confirmed an increase in transcription activity of AP1 to the UII promoter under hypoxia. After hypoxia, an increase in (3)H-proline incorporation in the cardiomyocytes and expression of myosin heavy chain protein, indicative of cardiomyocyte hypertrophy, were observed. In addition, hypoxia increased collagen I expression, which was inhibited by SP600125, NAC, and UII siRNA. In summary, hypoxia in cardiomyocytes increases UII and collagen I expression through the induction of AngII, ROS, and the JNK pathway causing cardiomyocyte hypertrophy and fibrosis.

Sodium Ferulate Inhibits Neointimal Hyperplasia in Rat Balloon Injury Model

Background/AimNeointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved.MethodsCultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed.ResultsIn presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group.ConclusionSodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs.

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.

DNA demethylation enhances myoblasts hypertrophy during the late phase of myogenesis activating the IGF-I pathway

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both physical activity and pathological stimuli. This research was performed to investigate DNA demethylation action on the late phase of muscle differentiation and early stage of hypertrophy. The epigenetic process involved in myogenesis was studied with the DNA-demethylating agent 5-azacytidine (AZA). We induced muscle differentiation in C2C12 mouse myoblasts in the presence of 5 μM AZA and growth or differentiation medium for 48, 72, and 96 h. To study a potential AZA hypertrophic effect, we stimulated 72 h differentiated myotubes with AZA for 24 h. Unstimulated cells were used as control. By western blot and immunofluorescence analysis, we examined AZA action on myogenic regulatory factors expression, hypertrophic signaling pathway and myotube morphology. During differentiation, protein levels of myogenic markers, Myf6 and Myosin Heavy Chain (MyHC), were higher in AZA stimulated cells compared to control. Myostatin and p21 analysis revealed morphological changes which reflect a tendency to hypertrophy in myotubes. In AZA stimulated neo formed myotubes, we observed that IGF-I pathway, kinases p70 S6, 4E-BP1, and ERK1/2 were activated. Furthermore, AZA treatment increased MyHC protein content in stimulated neo myotubes. Our work demonstrates that DNA demethylation could plays an important role in promoting the late phase of myogenesis, activating endocellular pathways involved in protein increment and stimulating the hypertrophic process.

Recessive myosin myopathy with external ophthalmoplegia associated with MYH2 mutations

Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild-to-moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe for the first time the clinical and morphological characteristics of recessive myosin IIa myopathy associated with MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild-to-moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated small or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that mild muscle weakness and ophthalmoplegia in combination with muscle biopsy demonstrating small or absent type 2A muscle fibers are the hallmark of recessive myopathy associated with MYH2 mutations.

CrossTalk proposal: Mechanical ventilation‐induced diaphragm atrophy is primarily due to inactivity

CrossTalk proposal: Mechanical ventilation‐induced diaphragm atrophy is primarily due to inactivity

Proteins degradation value in cured meat product made from M. Cutaneous-omo brachialis muscle of bovine

We analyzed differences in some physicochemical parameters of proteins in shoulder rose (M. Cutaneous-omo brachialis) muscle as a result of beef processing to produce pastirma. Samples from processed muscles showed significantly increased concentrations of extracted proteins, especially of H2O P-ex and Guba-Straub–adenosine triphosphate solution (P < 0.01), as a result of the salting–curing process. The salt-curing process was likely to have an important effect on the extractability of muscle proteins such as myosin heavy chain (MHC) and water-soluble proteins, possibly as a result of releasing some proteins from each other and cleaving the structures between certain proteins. The fluorescence intensities of processed samples were higher than those of the control samples at all guanidine hydrochloride concentrations. The hydrophobicity also increased on account of new compounds that were created during the pastirma-making process. Since the process of making pastirma lasts about 4 weeks (drying), the metmyoglobin content was greatly increased in pastirma samples compared with the unprocessed samples (by as much as 57 %). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results conceived that during processing of pastirma products, MHC and other muscle proteins degraded into polypeptides with smaller molecular weight lower than 15-kDa. The results of this study demonstrated that meat processing promoted the enzymatic digestion of some proteins, and the differences in composition between the control and pastirma samples were thus likely to be owing to protein degradation. The traditional pastirma-making process hence has no negative impact on the structure of the muscle and produces a firmer-textured.

EIF4EBP3L Acts as a Gatekeeper of TORC1 In Activity-Dependent Muscle Growth by Specifically Regulating Mef2ca Translational Initiation

Author SummaryMost genes are transcribed into mRNA and then translated into proteins that function in various cellular processes. Initiation of mRNA translation is thus a fundamental control point in gene expression. Working in a zebrafish model, we have found that muscle activity (or inactivity) can differentially regulate the translation of specific mRNAs and thereby control the growth of skeletal muscle. Emerging evidence suggests that control of translational initiation of particular mRNAs by an intracellular signaling pathway acting through TORC1 is a major regulator of cell growth and function. We show here that muscle activity both activates the TORC1 pathway and suppresses the expression of a downstream TORC1 target—the translational inhibitor eIF4EBP3L. This removes a brake on translation of certain mRNAs. Conversely, we show that muscle inactivity can up-regulate this translational inhibitor, thereby causing reduced translation of these mRNAs. One of the mRNAs targeted in this manner by eIF4EBP3L is Mef2ca, which encodes a transcription factor that promotes assembly of muscle contractile apparatus. Our work thus reveals a mechanism by which muscle growth can be differentially influenced depending on the context of muscle activity (or lack thereof). If this pathway operates in people, it may help explain how exercise regulates muscle growth and performance.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.

TheCompactMutation of Myostatin Causes a Glycolytic Shift in the Phenotype of Fast Skeletal Muscles

Myostatin is an important negative regulator of skeletal muscle growth. The hypermuscular Compact (Cmpt) mice carry a 12-bp natural mutation in the myostatin propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast-type tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed-type soleus (SOL) muscles of Cmpt and wild-type mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qPCR. Based on our results, all investigated muscles of Cmpt mice were significantly larger compared with that of wild-type mice, as characterized by fiber hyperplasia of different grades. Fiber hypertrophy was not present in TA; however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles were generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by a decreased number of IIX and IIA fibers; however, this was not the case for SOL muscles. In summary, despite the variances found in muscle cellularity between the different myostatin mutant mice, similar glycolytic shifts were observed in Cmpt fast muscles as in muscles from myostatin knockout mice.

P26 Placental stem villus arteries undergo dedifferentiation via reduced hydrogen sulfide production by cystathionine-γ-lyase upon exposure to oxidative stress

Introduction Intrauterine growth restriction (IUGR) is a leading cause of perinatal morbidity and mortality, and is often associated with abnormal umbilical artery Doppler waveforms indicative of increased vascular resistance. The increased resistance is thought to involve stem villus arteries (SVAs), but how it develops remains unknown. Oxidative stress following deficient maternal spiral artery conversion is believed to be the primary insult in unexplained cases of IUGR and pre-eclampsia. Cystathionine- γ -lyase (CSE) has been reported to be downregulated in SVAs during IUGR and pre-eclampsia [1] . Methods SVA explants from uncomplicated human pregnancies delivered by elective Caesarean section were subjected to oxidative stress in vitro by cycles of hypoxia-reoxygenation (HR), or to normoxia in the presence or absence of CSE inhibitor propargylglycine (10 mM) for 1 or 3 days. We additionally analysed the protein sequences obtained from the Human Protein Reference Database to look for potential cysteine changes in the vasculature. Results HR induced oxidative stress, and vascular smooth muscle (SM) dedifferentiation, with reduced expression of the contractile proteins and differentiation markers SM Myosin Heavy Chain and SM α -actin. SM dedifferentiation was accompanied by acquisition of a synthetic phenotype characterised by increased SM proliferation, and marked ECM remodelling, as evidenced by changes in collagen deposition. HR also reduced CSE expression in SVAs and produced a decline in H 2 S production of similar magnitude. CSE inhibition under normoxic conditions increased the level of oxidative stress in SVAs and produced changes in SM marker expression similar to those induced by HR. Under oxidative stress, changes in Hsp90 and catalase levels were paradoxically correlated with changes in CSE expression. Finally, protein sequences obtained from the Human Protein Reference Database revealed cysteine enrichment in vascular tissues compared to other tissues. This was due to the presence of a subset of cysteine-rich extracellular proteins, and suggests H 2 S may act via protein S-sulfhydration in SVAs, including on extracellular targets. Conclusions Our results show that oxidative stress can trigger, via CSE downregulation and reduced H 2 S signalling, a positive feedback loop of signalling that drives SM dedifferentiation into a synthetic phenotype. This could result in vascular remodelling, leading to compromised SVA distensibility, and hence increased SVA and placental resistance in pathological pregnancies.

Comparison of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Type Proportions in the Rat Slow and Fast Muscles

We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts (MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.

Oxidative stress induces smooth muscle dedifferentiation in placental stem villus arteries through reduced hydrogen sulphide production by cystathionine-γ-lyase

Introduction: Intrauterine growth restriction (IUGR) is a leading cause of perinatal morbidity and mortality, and is often associated with abnormal Doppler umbilical artery waveforms indicative of increased vascular resistance. The increased resistance is thought to involve stem villus arteries (SVAs), but how it develops remains unknown. Oxidative stress following deficient maternal spiral artery conversion is believed to be the primary insult in unexplained cases of IUGR and pre-eclampsia. Cystathionine-g-lyase (CSE) has been reported to be downregulated in SVAs during IUGR and pre-eclampsia1. Methods: SVA explants from uncomplicated human pregnancies delivered by elective Caesarean section were subjected to oxidative stress in vitro by cycles of hypoxia-reoxygenation (HR), or to normoxia in the presence or absence of CSE inhibitor propargylglycine (10 mM) for 1 or 3 days. Results: HR induced oxidative stress, and vascular smooth muscle (SM) dedifferentiation, with reduced expression of the contractile proteins and differentiation markers SM Myosin Heavy Chain and SM a-actin. SM dedifferentiation was accompanied by acquisition of a synthetic phenotype characterised by increased SM proliferation, and marked ECM remodelling, as evidenced by changes in collagen deposition. HR also reduced CSE expression in SVAs and produced a decline in H2S production of similar magnitude. CSE inhibition under normoxic conditions increased the level of oxidative stress in SVAs and produced changes in SM marker expression similar to those induced by HR. Under oxidative stress, changes in Hsp90 and catalase levels were paradoxically correlated with changes in CSE expression. Conclusions: Our results show that oxidative stress can trigger, via CSE downregulation and reduced H2S signalling, a positive feedback loop of signalling that drives SM dedifferentiation into a synthetic phenotype. This could result in vascular remodelling, leading to compromised SVA distensibility, and hence increased SVA and placental resistance in pathological pregnancies. 1. Cindrova-Davies et al. 2013 Am J Pathol 182,1448-1458.

Distinct effects of methamphetamine on autophagy–lysosome and ubiquitin–proteasome systems in HL-1 cultured mouse atrial cardiomyocytes

The aim of this study is to investigate the molecular mechanism underling the cardiotoxicity of methamphetamine, a psychostimulant drug that is currently abused in the world. A mouse atrial cardiac cell line, HL-1, which retains phenotypes of cardiac cells and serves as a useful model for examining cardiac pathophysiology, was used for this purpose. During treatment with 1mM methamphetamine (MAP) for 3–48h, massive but transient cytoplasmic vacuolization (3–12h) followed by an intracellular accumulation of granules (24–48h) was observed under light microscopy. The vacuoles were surrounded by the lysosome membrane marker LAMP1, while the granules colocalized with the autophagy markers LC3 and p62 as well as ubiquitinated proteins. Western blot analysis showed that LC3 was activated during MAP administration, although p62 was not degraded but rather accumulated. Concordant with p62 accumulation, the nuclear translocation of an anti-oxidative transcription factor, Nrf2, and the subsequent induction of its target gene, HO-1, was observed, suggesting an impairment of autophagic protein degradation and the subsequent activation of the p62/Nrf2/HO-1 pathway. In addition, proteomic analysis revealed a reduction in myosin heavy chain (MHC) protein levels during MAP administration. The ubiquitination of MHC and the induction of the muscle sarcomere protein-specific E3 ubiquitin ligases MuRF1 and atrogin-1 were proved by immunoprecipitation and quantitative real-time PCR, respectively. Taken together, the vacuolization of lysosomes and the subsequent accumulation of autophagosomes indicate an impairment of autophagic protein degradation during MAP administration; on the other hand, the ubiquitination and subsequent degradation of MHC indicate the proper progression of proteasomal degradation.