IIb or not IIb? Regulation of myosin heavy chain gene expression in mice and men

<p>Abstract</p> <p>Background</p> <p>While the myosin heavy chain IIb isoform (MyHC-IIb) is the predominant motor protein in most skeletal muscles of rats and mice, the messenger RNA (mRNA) for this isoform is only expressed in a very small subset of specialized muscles in adult large mammals, including humans.</p> <p>Results</p> <p>We identify the DNA sequences limiting MyHC-IIb expression in humans and explore the activation of this gene in human skeletal muscle. We demonstrate that the transcriptional activity of ~1.0 kb of the human MyHC-IIb promoter is greatly reduced compared to that of the corresponding mouse sequence in both mouse and human myotubes <it>in vitro </it>and show that nucleotide differences that eliminate binding sites for myocyte enhancer factor 2 (MEF2) and serum response factor (SRF) account for this difference. Despite these differences, we show that MyHC-IIb mRNA is expressed in fetal human muscle cells and that MyHC-IIb mRNA is significantly up-regulated in the skeletal muscle of Duchene muscular dystrophy patients.</p> <p>Conclusions</p> <p>These data identify the genetic basis for a key phenotypic difference between the muscles of large and small mammals, and demonstrate that mRNA expression of the MyHC-IIb gene can be re-activated in human limb muscle undergoing profound degeneration/regeneration.</p

A comparative perspective on three primate species’ responses to a pictorial emotional Stroop task

This study was also supported (in part) by a grant from The David Bohnett Foundation, the Leo S. Guthman Fund, the Chauncey and Marion Deering McCormick Foundation, and, at the time of writing, L.M.H. was supported by the Women’s Board of Lincoln Park Zoo.The Stroop effect describes interference in cognitive processing due to competing cognitive demands. Presenting emotionally laden stimuli creates similar Stroop-like effects that result from participants’ attention being drawn to distractor stimuli. Here, we adapted the methods of a pictorial Stroop study for use with chimpanzees (N = 6), gorillas (N = 7), and Japanese macaques (N = 6). We tested all subjects via touchscreens following the same protocol. Ten of the 19 subjects passed pre-test training. Subjects who reached criterion were then tested on a standard color-interference Stroop test, which revealed differential accuracy in the primates’ responses across conditions. Next, to test for an emotional Stroop effect, we presented subjects with photographs that were either positively valenced (a preferred food) or negatively valenced (snakes). In the emotional Stroop task, as predicted, the primates were less accurate in trials which presented emotionally laden stimuli as compared to control trials, but there were differences in the apes’ and monkeys’ response patterns. Furthermore, for both Stroop tests, while we found that subjects’ accuracy rates were reduced by test stimuli, in contrast to previous research, we found no difference across trial types in the subjects’ response latencies across conditions.Publisher PDFPeer reviewe

Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes. © 2006 Geurts et al

UK construction companies’ strategies in the face of business cycles

Firms in the construction industry have always had to deal with the challenges of the economic cycle and develop strategies to deal with the resulting fluctuations in their business environment. In the context of the 2008–2011 double-dip recession in the UK, the results of a survey targeting the top one hundred construction companies in the UK are reported here. This research is particularly intended to assess whether the strategies of large companies in the construction sector, when faced with the issues associated with the variation in the economic cycle, have changed since the previous business cycle (i.e. the 1986–1990 boom followed by the 1990–1991 recession). The survey reveals the challenges that companies have faced, reports on company behaviour and on the policies adopted. While there are many similarities between policies adopted during the recessionary periods of the two cycles, the research found notable changes in attitudes towards diversification, human resource management and price bidding

The Role of MEKK1 in Hypertrophic Cardiomyopathy

MEKK1 is a ubiquitously expressed mitogen activated protein kinase that is involved in tissue remodeling in a variety of settings including carotid artery blood flow cessation, wound healing and breast adenocarcinoma intravasation. Here, we have tested the function of MEKK1 in genetic hypertrophic cardiomyopathy (HCM). MEKK1 was genetically deleted in C57Bl6/J mice expressing a mutant β-myosin heavy chain (HCM-MEKK1-/-). The absence of MEKK1 in HCM resulted in a more pronounced hypertrophy when compared to HCM mice with the MEKK1 gene intact without further increases in atrial natriuretic factor and β-myosin heavy chain (MyHC) expression and fibrosis. Since MEKK1 is required for the induction of several tissue proteases, we tested the hypothesis that cardiac enlargement of HCM- MEKK1-/- mice was due to altered expression of urokinase-type plasminogen activator (uPA), JunB, matrix-metalloproteinase (MMP), and tissue inhibitors of MMPs (TIMPs). Because of its role in preventing apoptosis, we also tested the loss of MEKK1 on apoptotic mediators Bcl-2, cytochrome C, caspase-9 and caspase-3. uPA expression was decreased while Jun B, MMP-9, caspase-9 and caspase-3 activities were elevated in HCM- MEKK1-/- hearts when compared to MEKK1-/-, wild-type (WT) and HCM mice. Bcl-2 and Cyt C expression was elevated only in HCM mice. We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis

Cover Image Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment

Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGF&beta;1 (transforming growth factor &beta; 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGF&beta;1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGF&beta;1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGF&beta;1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGF&beta;1 caused myocyte hypertrophy, most likely through insulin growth factor 1.</p

Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to beta-Adrenergic Stimulation

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic &beta;‐adrenergic receptor (&beta;‐AR)‐mediated cardiovascular disease. ISO‐treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, &beta;‐AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of &beta;1‐ and &beta;2‐ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO‐treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo &beta;‐AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher &beta;‐AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury. </div

Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding