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Find video protocols related to scientific articles indexed in Pubmed.
Targeting latent TGF? release in muscular dystrophy.
Sci Transl Med
PUBLISHED: 10-24-2014
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Latent transforming growth factor-? (TGF?) binding proteins (LTBPs) bind to inactive TGF? in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGF? release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGF? signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGF? release and activity and decrease inflammation and muscle damage in muscular dystrophy.
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Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy.
Circ Cardiovasc Genet
PUBLISHED: 09-01-2014
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-Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy.
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Myofiber-specific inhibition of TGF? signaling protects skeletal muscle from injury and dystrophic disease in mice.
Hum. Mol. Genet.
PUBLISHED: 08-08-2014
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Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-? has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGF?-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGF? signaling owing to expression of a TGF? type II receptor dominant-negative (dnTGF?RII) truncation mutant. Expression of dnTGF?RII in myofibers mitigated the dystrophic phenotype observed in ?-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGF?RII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGF?RII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGF? directly inhibited Mt gene expression in vitro, the dnTGF?RII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGF? signaling in MD.
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Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy.
Hum. Mol. Genet.
PUBLISHED: 07-28-2014
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Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) ? signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGF? signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein ?-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGF? since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGF? signaling alters cardiac and muscle performance through the intracellular SMAD pathway.
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P38? MAPK underlies muscular dystrophy and myofiber death through a Bax-dependent mechanism.
Hum. Mol. Genet.
PUBLISHED: 05-29-2014
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Muscular dystrophies are a group of genetic diseases that lead to muscle wasting and, in most cases, premature death. Cytokines and inflammatory factors are released during the disease process where they promote deleterious signaling events that directly participate in myofiber death. Here, we show that p38?, a kinase in the greater mitogen-activated protein kinase (MAPK)-signaling network, serves as a nodal regulator of disease signaling in dystrophic muscle. Deletion of Mapk14 (p38?-encoding gene) in the skeletal muscle of mdx- (lacking dystrophin) or sgcd- (?-sarcoglycan-encoding gene) null mice resulted in a significant reduction in pathology up to 6 months of age. We also generated MAPK kinase 6 (MKK6) muscle-specific transgenic mice to model heightened p38? disease signaling that occurs in dystrophic muscle, which resulted in severe myofiber necrosis and many hallmarks of muscular dystrophy. Mechanistically, we show that p38? directly induces myofiber death through a mitochondrial-dependent pathway involving direct phosphorylation and activation of the pro-death Bcl-2 family member Bax. Indeed, muscle-specific deletion of Bax, but not the apoptosis regulatory gene Tp53 (encoding p53), significantly reduced dystrophic pathology in the muscles of MKK6 transgenic mice. Moreover, use of a p38 MAPK pharmacologic inhibitor reduced dystrophic disease in Sgcd(-/-) mice suggesting a future therapeutic approach to delay disease.
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Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-09-2014
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Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury.
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Exon-skipping therapy: a roadblock, detour, or bump in the road?
Sci Transl Med
PUBLISHED: 04-04-2014
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Exon skipping is a promising therapeutic for Duchenne muscular dystrophy patients, but the road to drug approvals is foggy and may require more early-stage derisking and regulatory guidance.
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Abcc9 is required for the transition to oxidative metabolism in the newborn heart.
FASEB J.
PUBLISHED: 03-19-2014
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The newborn heart adapts to postnatal life by shifting from a fetal glycolytic metabolism to a mitochondrial oxidative metabolism. Abcc9, an ATP-binding cassette family member, increases expression concomitant with this metabolic shift. Abcc9 encodes a membrane-associated receptor that partners with a potassium channel to become the major potassium-sensitive ATP channel in the heart. Abcc9 also encodes a smaller protein enriched in the mitochondria. We now deleted exon 5 of Abcc9 to ablate expression of both plasma membrane and mitochondria-associated Abcc9-encoded proteins, and found that the myocardium failed to acquire normal mature metabolism, resulting in neonatal cardiomyopathy. Unlike wild-type neonatal cardiomyocytes, mitochondria from Ex5 cardiomyocytes were unresponsive to the KATP agonist diazoxide, consistent with loss of KATP activity. When exposed to hydrogen peroxide to induce cell stress, Ex5 neonatal cardiomyocytes displayed a rapid collapse of mitochondria membrane potential, distinct from wild-type cardiomyocytes. Ex5 cardiomyocytes had reduced fatty acid oxidation, reduced oxygen consumption and reserve. Morphologically, Ex5 cardiac mitochondria exhibited an immature pattern with reduced cross-sectional area and intermitochondrial contacts. In the absence of Abcc9, the newborn heart fails to transition normally from fetal to mature myocardial metabolism.-Fahrenbach, J. P., Stoller, D., Kim, G., Aggarwal, N., Yerokun, B., Earley, J. U., Hadhazy, M., Shi, N.-Q., Makielski, J. C., McNally, E. M. Abcc9 is required for the transition to oxidative metabolism in the newborn heart.
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GRAF1 promotes ferlin-dependent myoblast fusion.
Dev. Biol.
PUBLISHED: 03-12-2014
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Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.
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Dynamin 2 the rescue for centronuclear myopathy.
J. Clin. Invest.
PUBLISHED: 02-24-2014
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Centronuclear myopathy is a lethal muscle disease. The most severe form of the disease, X-linked centronuclear myopathy, is due to mutations in the gene encoding myotubularin (MTM1), while mutations in dynamin 2 (DNM2) and amphiphysin 2/BIN1 (AMPH2) cause milder forms of myopathy. MTM1 is a lipid phosphatase, and mutations that disrupt this activity cause severe muscle wasting. In this issue of the JCI, Cowling and colleagues report on their finding of increased DNM2 levels in human and mouse muscle with MTM1 mutations. Partial reduction of Dnm2 in mice harboring Mtm1 mutations remarkably rescued muscle wasting and lethality, and this effect was muscle specific. DNM2 regulates membrane trafficking through vesicular scission, and it is presumed that reducing this activity accounts for improved outcome in X-linked centronuclear myopathy.
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Supercomputing for the parallelization of whole genome analysis.
Bioinformatics
PUBLISHED: 02-12-2014
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The declining cost of generating DNA sequence is promoting an increase in whole genome sequencing, especially as applied to the human genome. Whole genome analysis requires the alignment and comparison of raw sequence data, and results in a computational bottleneck because of limited ability to analyze multiple genomes simultaneously.
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Genetic profiling for risk reduction in human cardiovascular disease.
Genes (Basel)
PUBLISHED: 01-16-2014
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Cardiovascular disease is a major health concern affecting over 80,000,000 people in the U.S. alone. Heart failure, cardiomyopathy, heart rhythm disorders, atherosclerosis and aneurysm formation have significant heritable contribution. Supported by familial aggregation and twin studies, these cardiovascular diseases are influenced by genetic variation. Family-based linkage studies and population-based genome-wide association studies (GWAS) have each identified genes and variants important for the pathogenesis of cardiovascular disease. The advent of next generation sequencing has ushered in a new era in the genetic diagnosis of cardiovascular disease, and this is especially evident when considering cardiomyopathy, a leading cause of heart failure. Cardiomyopathy is a genetically heterogeneous disorder characterized by morphologically abnormal heart with abnormal function. Genetic testing for cardiomyopathy employs gene panels, and these panels assess more than 50 genes simultaneously. Despite the large size of these panels, the sensitivity for detecting the primary genetic defect is still only approximately 50%. Recently, there has been a shift towards applying broader exome and/or genome sequencing to interrogate more of the genome to provide a genetic diagnosis for cardiomyopathy. Genetic mutations in cardiomyopathy offer the capacity to predict clinical outcome, including arrhythmia risk, and genetic diagnosis often provides an early window in which to institute therapy. This discussion is an overview as to how genomic data is shaping the current understanding and treatment of cardiovascular disease.
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EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.
Dev. Biol.
PUBLISHED: 01-06-2014
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EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.
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The CO-Regulation Database (CORD): a tool to identify coordinately expressed genes.
PLoS ONE
PUBLISHED: 01-01-2014
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Meta-analysis of gene expression array databases has the potential to reveal information about gene function. The identification of gene-gene interactions may be inferred from gene expression information but such meta-analysis is often limited to a single microarray platform. To address this limitation, we developed a gene-centered approach to analyze differential expression across thousands of gene expression experiments and created the CO-Regulation Database (CORD) to determine which genes are correlated with a queried gene.
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Modifiers of Heart and Muscle Function: Where Genetics Meets Physiology.
Exp. Physiol.
PUBLISHED: 11-08-2013
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Many single gene disorders are associated with a range of symptoms that cannot be solely explained by the primary genetic mutation. Muscular dystrophy is a genetic disorder associated with variable outcomes that arises from both the primary genetic mutation and the contribution from environmental and genetic modifiers. Disruption of the dystrophin complex occurs in Duchenne muscular dystrophy and limb girdle muscular dystrophy producing heart and muscle disease through a cellular injury process characterized by plasma membrane disruption and fibrosis. Multiple modifier loci have been mapped by using a mouse model of muscular dystrophy. These modifiers exert their effect often on specific muscle groups targeted by the muscular dystrophy process, possibly reflecting distinct pathophysiological processes among muscle groups. Genetic modifiers act on both cardiac and respiratory muscle parameters suggesting genetic and physiological integration of cardiopulmonary function. Skeletal muscles of the limbs are modified by a locus on mouse chromosome 7. This region of chromosome 7 harbors an insertion/deletion polymorphism in Ltbp4, the gene encoding latent TGF? binding protein. Ltbp4 exerts its effect in muscle disease by acting on plasma membrane stability and fibrosis, thereby linking instability of the sarcolemma directly to fibrosis. In the human muscle disease Duchenne Muscular Dystrophy, protein coding single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation demonstrating that modifiers identified from mouse studies translate to human disease.
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Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity.
Am. J. Pathol.
PUBLISHED: 04-02-2013
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Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.
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Scanning transmission electron microscopic tomography of cortical bone using Z-contrast imaging.
Micron
PUBLISHED: 02-19-2013
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Previously we presented (McNally et al., 2012) a model for the ultrastructure of bone showing that the mineral resides principally outside collagen fibrils in the form of 5 nm thick mineral structures hundreds of nanometers long oriented parallel to the fibrils. Here we use high-angle annular dark-field electron tomography in the scanning transmission electron microscope to confirm this model and further elucidate the composite structure. Views of a section cut parallel to the fibril axes show bundles of mineral structures extending parallel to the fibrils and encircling them. The mineral density inside the fibrils is too low to be visualized in these tomographic images. A section cut perpendicular to the fibril axes, shows quasi-circular walls composed of mineral structures, wrapping around apparently empty holes marking the sites of fibrils. These images confirm our original model that the majority of mineral in bone resides outside the collagen fibrils.
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Modifying muscular dystrophy through transforming growth factor-?.
FEBS J.
PUBLISHED: 01-06-2013
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Muscular dystrophy arises from ongoing muscle degeneration and insufficient regeneration. This imbalance leads to loss of muscle, with replacement by scar or fibrotic tissue, resulting in muscle weakness and, eventually, loss of muscle function. Human muscular dystrophy is characterized by a wide range of disease severity, even when the same genetic mutation is present. This variability implies that other factors, both genetic and environmental, modify the disease outcome. There has been an ongoing effort to define the genetic and molecular bases that influence muscular dystrophy onset and progression. Modifier genes for muscle disease have been identified through both candidate gene approaches and genome-wide surveys. Multiple lines of experimental evidence have now converged on the transforming growth factor-? (TGF-?) pathway as a modifier for muscular dystrophy. TGF-? signaling is upregulated in dystrophic muscle as a result of a destabilized plasma membrane and/or an altered extracellular matrix. Given the important biological role of the TGF-? pathway, and its role beyond muscle homeostasis, we review modifier genes that alter the TGF-? pathway and approaches to modulate TGF-? activity to ameliorate muscle disease.
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Genetic mutations and mechanisms in dilated cardiomyopathy.
J. Clin. Invest.
PUBLISHED: 01-02-2013
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Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of congestive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are "private" or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with mutations in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is characterized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia.
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Vasodilation induced by oxygen/glucose deprivation is attenuated in cerebral arteries of SUR2 null mice.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 07-22-2011
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Physiological functions of arterial smooth muscle cell ATP-sensitive K(+) (K(ATP)) channels, which are composed of inwardly rectifying K(+) channel 6.1 and sulfonylurea receptor (SUR)-2 subunits, during metabolic inhibition are unresolved. In the present study, we used a genetic model to investigate the physiological functions of SUR2-containing K(ATP) channels in mediating vasodilation to hypoxia, oxygen and glucose deprivation (OGD) or metabolic inhibition, and functional recovery following these insults. Data indicate that SUR2B is the only SUR isoform expressed in murine cerebral artery smooth muscle cells. Pressurized SUR2 wild-type (SUR2(wt)) and SUR2 null (SUR2(nl)) mouse cerebral arteries developed similar levels of myogenic tone and dilated similarly to hypoxia (<10 mmHg Po(2)). In contrast, vasodilation induced by pinacidil, a K(ATP) channel opener, was ?71% smaller in SUR2(nl) arteries. Human cerebral arteries also expressed SUR2B, developed myogenic tone, and dilated in response to hypoxia and pinacidil. OGD, oligomycin B (a mitochondrial ATP synthase blocker), and CCCP (a mitochondrial uncoupler) all induced vasodilations that were ?39-61% smaller in SUR2(nl) than in SUR2(wt) arteries. The restoration of oxygen and glucose following OGD or removal of oligomycin B and CCCP resulted in partial recovery of tone in both SUR2(wt) and SUR2(nl) cerebral arteries. However, SUR(nl) arteries regained ?60-82% more tone than did SUR2(wt) arteries. These data indicate that SUR2-containing K(ATP) channels are functional molecular targets for OGD, but not hypoxic, vasodilation in cerebral arteries. In addition, OGD activation of SUR2-containing K(ATP) channels may contribute to postischemic loss of myogenic tone.
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Distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or the sarcoglycan complex.
FASEB J.
PUBLISHED: 06-10-2011
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Duchenne muscular dystrophy (DMD) and limb girdle muscular dystrophy (LGMD) 2C-F result from the loss of dystrophin and the sarcoglycans, respectively. Dystrophin, a cytoskeletal protein, is closely associated with the membrane-bound sarcoglycan complex. Despite this tight biochemical association, the function of dystrophin and the sarcoglycan subunits may differ. The loss of dystrophin in skeletal muscle results in muscle that is highly susceptible to contraction-induced damage, but the skeletal muscle of mice lacking ?- or ?-sarcoglycan are less susceptible. Using mouse models of DMD, LGMD-2C, and LGMD-2F, we demonstrate that isolated cardiac myocytes from mice lacking either ?- or ?-sarcoglycan have normal compliance. In contrast, dystrophin-deficient myocytes display poor passive compliance and are susceptible to terminal contracture following mild passive extensions. Mice deficient in dystrophin and, less so, ?-sarcoglycan have reduced survival during in vivo dobutamine stress testing compared to controls. Catheter-based hemodynamic studies show deficits in both baseline and dobutamine-stimulated cardiac function in all of the dystrophic mice compared to control mice, with dystrophin-deficient mice having the poorest function. In contrast, histopathology showed increased fibrosis in the sarcoglycan-deficient hearts, but not in hearts lacking dystrophin. In summary, this study provides important insights into the unique mechanisms of disease underlying these different models of inherited dystrophic cardiomyopathy and supports a model where dystrophin, but not the sarcoglycans, protects the cardiac myocyte against mechanical damage.
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Ferlin proteins in myoblast fusion and muscle growth.
Curr. Top. Dev. Biol.
PUBLISHED: 05-31-2011
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Myoblast fusion contributes to muscle growth in development and during regeneration of mature muscle. Myoblasts fuse to each other as well as to multinucleate myotubes to enlarge the myofiber. The molecular mechanisms of myoblast fusion are incompletely understood. Adhesion, apposition, and membrane fusion are accompanied by cytoskeletal rearrangements. The ferlin family of proteins is implicated in human muscle disease and has been implicated in fusion events in muscle, including myoblast fusion, vesicle trafficking and membrane repair. Dysferlin was the first mammalian ferlin identified and it is now known that there are six different ferlins. Loss-of-function mutations in the dysferlin gene lead to limb girdle muscular dystrophy and the milder disorder Miyoshi Myopathy. Dysferlin is a membrane-associated protein that has been implicated in resealing disruptions in the muscle plasma membrane. Newer data supports a broader role for dysferlin in intracellular vesicular movement, a process also important for resealing. Myoferlin is highly expressed in myoblasts that undergoing fusion, and the absence of myoferlin leads to impaired myoblast fusion. Myoferlin also regulates intracellular trafficking events, including endocytic recycling, a process where internalized vesicles are returned to the plasma membrane. The trafficking role of ferlin proteins is reviewed herein with a specific focus as to how this machinery alters myogenesis and muscle growth.
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Emery-Dreifuss muscular dystrophy.
Handb Clin Neurol
PUBLISHED: 04-19-2011
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Emery-Dreifuss muscular dystrophy (EDMD) is a progressive muscle-wasting disorder defined by early contractures of the Achilles tendon, spine, and elbows. EDMD is also distinctive for its association with defects of the cardiac conduction system that can result in sudden death. It can be inherited in an X-linked, autosomal dominant, or autosomal recessive fashion and is caused by mutations in proteins of the nuclear membrane. Mutations in the EMD gene, which encodes emerin, a transmembrane protein found at the inner nuclear membrane, are responsible for X-linked EDMD. The most common etiology of autosomal dominant EDMD is an LMNA gene mutation; LMNA encodes the intermediate filament protein lamins A and C, which constitute the major scaffolding protein of the inner nuclear membrane. Murine models of LMNA gene mutations have helped to identify different mechanisms of disease. Loss of LMNA function leads to nuclear fragility as well as other defects, such as abnormal nuclear function. Additional genes encoding nuclear membrane proteins such as SYNE1 and SYNE2 have also been implicated in EDMD, and in some cases their importance for cardiac and muscle function has been supported by animal modeling.
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Gene expression, chromosome position and lamin A/C mutations.
Nucleus
PUBLISHED: 03-31-2011
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The nuclear lamina is increasingly being appreciated for its epigenetic role in regulating gene expression. The nuclear lamina underlies the inner nuclear membrane and, in post mitotic cells, is composed of a latticework primarily formed by the intermediate filament protein, lamin A/C. Although not well defined, lamin-associated domains have been described, and these domains are determined by DNA sequence and chromatin conformation. Lamin-associated domains are positioned to mediate the interaction with the nuclear membrane, where they contribute to transcriptional regulation. Although lamin-associated domains are primarily considered to be repressive in nature, those nearer to nuclear pores may actually promote transcription. Mutations in LMNA, the gene encoding lamins A and C, are a relatively common cause of inherited cardiomyopathy. As substantial data supports a role for the lamina in its interaction with chromatin and gene regulation, we examined the role of a genetically disrupted lamina and the consequences thereof. A dominant LMNA mutation, E161K, that causes inherited cardiomyopathy was studied. Gene expression changes were profiled in a human cardiomyopathic E161K heart, and it was found that chromosome 13 had a high percentage of misexpressed genes. Chromosome 13 was also found to be less tightly associated with the nuclear membrane in E161K mutant cells, thereby linking abnormal gene expression and intranuclear position. These and other studies support a role for the nuclear membrane as an active regulator of gene expression and provide additional support that disrupting this regulation is a mechanism of human disease.
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Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane KATP channels.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 03-18-2011
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Hydrogen sulfide (H(2)S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H(2)S exist. Here, we tested the hypothesis that piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K(+) (K(ATP)) currents and that H(2)S induces vasodilation by activating K(ATP) currents. Gas chromatography/mass spectrometry data demonstrated that after placing Na(2)S, an H(2)S donor, in solution, it rapidly (1 min) converts to H(2)S. Patch-clamp electrophysiology indicated that pinacidil (a K(ATP) channel activator), Na(2)S, and NaHS (another H(2)S donor) activated K(+) currents at physiological steady-state voltage (-50 mV) in isolated cerebral arteriole smooth muscle cells. Glibenclamide, a selective K(ATP) channel inhibitor, fully reversed pinacidil-induced K(+) currents and partially reversed (?58%) H(2)S-induced K(+) currents. Western blot analysis indicated that piglet arterioles expressed inwardly rectifying K(+) 6.1 (K(ir)6.1) channel and sulfonylurea receptor 2B (SUR2B) K(ATP) channel subunits. Pinacidil dilated pressurized (40 mmHg) piglet arterioles, and glibenclamide fully reversed this effect. Na(2)S also induced reversible and repeatable vasodilation with an EC(50) of ?30 ?M, and this effect was partially reversed (?55%) by glibenclamide. Vasoregulation by H(2)S was also studied in pressurized resistance-size cerebral arteries of mice with a genetic deletion in the gene encoding SUR2 (SUR2 null). Pinacidil- and H(2)S-induced vasodilations were smaller in arterioles of SUR2 null mice than in wild-type controls. These data indicate that smooth muscle cell K(ATP) currents control newborn cerebral arteriole contractility and that H(2)S dilates cerebral arterioles by activating smooth muscle cell K(ATP) channels containing SUR2 subunits.
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The emerging genetic landscape underlying cardiac conduction system function.
Birth Defects Res. Part A Clin. Mol. Teratol.
PUBLISHED: 01-26-2011
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Proper function of an organized Cardiac Conduction System (CCS) is vital to the survival of metazoans ranging from fly to man. The routine use of non-invasive electrocardiogram measures in the diagnosis and monitoring of cardiovascular health has established a trove of reliable CCS functional data in both normal and diseased cardiac states. Recent combination of echocardiogram (ECG) data with genome-wide association studies has identified genomic regions implicated in ECG variability which impact CCS function. In this study, we review the substantial recent progress in this area, highlighting the identification of novel loci, confirming the importance of previously implicated loci in CCS function, and exploring potential links between genes with important roles in developmental processes and variation in function of the CCS.
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Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.
J. Biol. Chem.
PUBLISHED: 12-22-2010
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The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.
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SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy.
Hum. Mol. Genet.
PUBLISHED: 12-06-2010
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Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-? (TGF?) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGF? signaling is pathogenic in the muscle itself. Drosophila deleted for the ?/?-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. We used dad-lacZ flies to demonstrate the signature of TGF? activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGF? signaling. To determine the pathogenic nature of this signaling, we found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrected both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restored muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction.
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Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.
Hum. Mol. Genet.
PUBLISHED: 12-01-2010
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Loss-of-function mutations in dysferlin cause muscular dystrophy, and dysferlin has been implicated in resealing membrane disruption in myofibers. Given the importance of membrane fusion in many aspects of muscle function, we studied the role of dysferlin in muscle growth. We found that dysferlin null myoblasts have a defect in myoblast-myotube fusion, resulting in smaller myotubes in culture. In vivo, dysferlin null muscle was found to have mislocalized nuclei and vacuolation. We found that myoblasts isolated from dysferlin null mice accumulate enlarged, lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes. Dysferlin null myoblasts accumulate transferrin-488, reflecting abnormal vesicular trafficking. Additionally, dysferlin null myoblasts display abnormal trafficking of the insulin-like growth factor (IGF) receptor, where the receptor is shuttled to LAMP2-positive lysosomes. We studied growth, in vivo, by infusing mice with the growth stimulant IGF1. Control IGF1-treated mice increased myofiber diameter by 30% as expected, whereas dysferlin null muscles had no response to IGF1, indicating a defect in myofiber growth. We also noted that dysferlin null fibroblasts also accumulate acidic vesicles, IGF receptor and transferrin, indicating that dysferlin is important for nonmuscle vesicular trafficking. These data implicate dysferlin in multiple membrane fusion events within the cell and suggest multiple pathways by which loss of dysferlin contributes to muscle disease.
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S100A12 in vascular smooth muscle accelerates vascular calcification in apolipoprotein E-null mice by activating an osteogenic gene regulatory program.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 10-21-2010
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The proinflammatory cytokine S100A12 is associated with coronary atherosclerotic plaque rupture. We previously generated transgenic mice with vascular smooth muscle-targeted expression of human S100A12 and found that these mice developed aortic aneurysmal dilation of the thoracic aorta. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in atherosclerosis-prone apolipoprotein E (ApoE)-null mice would accelerate atherosclerosis.
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Distinct genetic regions modify specific muscle groups in muscular dystrophy.
Physiol. Genomics
PUBLISHED: 10-19-2010
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Phenotypic expression in the muscular dystrophies is variable, even with the identical mutation, providing strong evidence that genetic modifiers influence outcome. To identify genetic modifier loci, we used quantitative trait locus mapping in two differentially affected mouse strains with muscular dystrophy. Using the Sgcg model of limb girdle muscular dystrophy that lacks the dystrophin-associated protein ?-sarcoglycan, we evaluated chromosomal regions that segregated with two distinct quantifiable characteristics of muscular dystrophy, membrane permeability and fibrosis. We previously identified a single major locus on murine chromosome 7 that influences both traits of membrane permeability and fibrosis in the quadriceps muscle. Using a larger cohort, we now found that this same interval strongly associated with both traits in all limb skeletal muscle groups studied, including the gastrocnemius/soleus, gluteus/hamstring, and triceps muscles. In contrast, the muscles of the trunk were modified by distinct genetic loci, possibly reflecting the embryological origins and physiological stressors unique to these muscle groups. A locus on chromosome 18 was identified that modified membrane permeability of the abdominal muscles, and a locus on chromosome 3 was found that regulated diaphragm and abdominal muscle fibrosis. Fibrosis in the heart associated with a region on chromosome 9 and likely reflects differential function between cardiac and skeletal muscle. These data underscore the complexity of inheritance and penetrance of single-gene disorders.
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The mitochondrial bioenergetic phenotype for protection from cardiac ischemia in SUR2 mutant mice.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 10-08-2010
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The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K(ATP)) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K(ATP), either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (??(m)), tolerance to Ca(2+) load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K(+) were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ??(m) was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca(2+) loading was increased (163 ± 2 vs. 116 ± 2 ?M), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K(+) medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K(+) influx. Additionally, ??(m) decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ??(m) maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K(+) conductance that partially dissipated ??(m). These results have implications for possible SUR2 participation in mitochondrial K(ATP).
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Cardiac assessment in duchenne and becker muscular dystrophies.
Curr Heart Fail Rep
PUBLISHED: 09-22-2010
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Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophies. In addition to muscle disease, there nearly always is an associated cardiomyopathy in Duchenne or Becker muscular dystrophy. In these muscular dystrophies, the severity of cardiomyopathy and congestive heart failure may not parallel the severity of skeletal muscle disease. Loss of normal dystrophin function in the heart produces four-chamber dilation and reduction in left ventricular function that develop after the onset of muscle weakness. Arrhythmias affecting both atrial and ventricular rhythms occur and may be life threatening. The degree to which hypoventilation and pulmonary dysfunction are present also directly affect cardiac function in muscular dystrophy. Care guidelines recently were issued to outline surveillance and treatment strategies for the younger patient with Duchenne muscular dystrophy. Herein, we review those guidelines, and additionally, provide recommendations for monitoring and treating cardiac disease in the populations of advanced Duchenne and Becker muscular dystrophies.
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Genetic deletion of NOS3 increases lethal cardiac dysfunction following mouse cardiac arrest.
Resuscitation
PUBLISHED: 08-23-2010
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Cardiac arrest mortality is significantly affected by failure to obtain return of spontaneous circulation (ROSC) despite cardiopulmonary resuscitation (CPR). Severe myocardial dysfunction and cardiovascular collapse further affects mortality within hours of initial ROSC. Recent work suggests that enhancement of nitric oxide (NO) signaling within minutes of CPR can improve myocardial function and survival. We studied the role of NO signaling on cardiovascular outcomes following cardiac arrest and resuscitation using endothelial NO synthase knockout (NOS3(-/-)) mice.
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Cardiomyocyte sulfonylurea receptor 2-KATP channel mediates cardioprotection and ST segment elevation.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 07-23-2010
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Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.
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Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.
PLoS ONE
PUBLISHED: 07-19-2010
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Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.
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Myoferlin regulation by NFAT in muscle injury, regeneration and repair.
J. Cell. Sci.
PUBLISHED: 06-22-2010
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Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.
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The genetics of dilated cardiomyopathy.
Curr. Opin. Cardiol.
PUBLISHED: 02-27-2010
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More than 40 different individual genes have been implicated in the inheritance of dilated cardiomyopathy. For a subset of these genes, mutations can lead to a spectrum of cardiomyopathy that extends to hypertrophic cardiomyopathy and left ventricular noncompaction. In nearly all cases, there is an increased risk of arrhythmias. With some genetic mutations, extracardiac manifestations are likely to be present. The precise genetic cause can usually not be discerned from the cardiac and/or extracardiac manifestations and requires molecular genetic diagnosis for prognostic determination and cardiac care.
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Nesprins, but not sun proteins, switch isoforms at the nuclear envelope during muscle development.
Dev. Dyn.
PUBLISHED: 01-29-2010
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Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin-1-giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin-2 partly replaced nesprin-1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin-negative skin fibroblasts, nesprin-2-giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin-1 remained at the nuclear envelope. In emerin-negative keratinocytes lacking nesprin-1, nesprin-2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin-1, which was the dominant form in myoblasts, while a novel 130-kD nesprin-2 isoform dominated Ntera-2 cells. The results suggest the possibility of isoform-specific and tissue-specific roles for nesprins in nuclear positioning.
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Myoferlin is required for insulin-like growth factor response and muscle growth.
FASEB J.
PUBLISHED: 12-11-2009
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Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.-Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth.
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S100A12 mediates aortic wall remodeling and aortic aneurysm.
Circ. Res.
PUBLISHED: 10-29-2009
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S100A12 is a small calcium binding protein that is a ligand of RAGE (receptor for advanced glycation end products). RAGE has been extensively implicated in inflammatory states such as atherosclerosis, but the role of S100A12 as its ligand is less clear.
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Genetic manipulation of dysferlin expression in skeletal muscle: novel insights into muscular dystrophy.
Am. J. Pathol.
PUBLISHED: 10-15-2009
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Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca(2+)-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle-specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf(-/-) A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex-lacking Scgd (delta-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.
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Molecular identification and functional characterization of a mitochondrial sulfonylurea receptor 2 splice variant generated by intraexonic splicing.
Circ. Res.
PUBLISHED: 10-01-2009
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Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel but its composition is not fully understood.
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Mechanisms of muscle degeneration, regeneration, and repair in the muscular dystrophies.
Annu. Rev. Physiol.
PUBLISHED: 09-23-2009
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To withstand the rigors of contraction, muscle fibers have specialized protein complexes that buffer against mechanical stress and a multifaceted repair system that is rapidly activated after injury. Genetic studies first identified the mechanosensory signaling network that connects the structural elements of muscle and, more recently, have identified repair elements of muscle. Defects in the genes encoding the components of these systems lead to muscular dystrophy, a family of genetic disorders characterized by progressive muscle wasting. Although the age of onset, affected muscles, and severity vary considerably, all muscular dystrophies are characterized by muscle necrosis that overtakes the regenerative capacity of muscle. The resulting replacement of muscle by fatty and fibrous tissue leaves muscle increasingly weak and nonfunctional. This review discusses the cellular mechanisms that are primarily and secondarily disrupted in muscular dystrophy, focusing on membrane degeneration, muscle regeneration, and the repair of muscle.
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Impaired exercise tolerance and skeletal muscle myopathy in sulfonylurea receptor-2 mutant mice.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
PUBLISHED: 08-12-2009
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By sensing intracellular energy levels, ATP-sensitive potassium (K(ATP)) channels help regulate vascular tone, glucose metabolism, and cardioprotection. SUR2 mutant mice lack full-length K(ATP) channels in striated and smooth muscle and display a complex phenotype of hypertension and coronary vasospasm. SUR2 mutant mice also display baseline cardioprotection and can withstand acute sympathetic stress better than normal mice. We now studied response to a form of chronic stress, namely that induced by 4 wk of daily exercise on SUR2 mutant mice. Control mice increased exercise capacity by 400% over the training period, while SUR2 mutant mice showed little increase in exercise capacity. Unexercised SUR2 mutant showed necrotic and regenerating fibers in multiple muscle skeletal muscles, including quadriceps, tibialis anterior, and diaphragm muscles. Unlike exercised control animals, SUR2 mutant mice did not lose weight, presumably due to less overall exertion. Unexercised SUR2 mutant mice showed a trend of mildly reduced cardiac function, measured by fractional shortening, (46 +/- 4% vs. 57 +/- 7% for SUR2 mutant and control, respectively), and this decrease was not exacerbated by chronic exercise exposure. Despite an improved response to acute sympathetic stress and baseline cardioprotection, exercise intolerance results from lack of SUR2 K(ATP) channels in mice.
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Nesprin-1 mutations in human and murine cardiomyopathy.
J. Mol. Cell. Cardiol.
PUBLISHED: 07-29-2009
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Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.
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Sarcomere mutations in cardiomyopathy with left ventricular hypertrabeculation.
Circ Cardiovasc Genet
PUBLISHED: 07-24-2009
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Mutations in the genes encoding sarcomere proteins have been associated with both hypertrophic and dilated cardiomyopathy. Recently, mutations in myosin heavy chain (MYH7), cardiac actin (ACTC), and troponin T (TNNT2) were associated with left ventricular noncompaction, a form of cardiomyopathy characterized with hypertrabeculation that may also include reduced function of the left ventricle.
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Mutation of SYNE-1, encoding an essential component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis.
Hum. Mol. Genet.
PUBLISHED: 06-19-2009
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Arthrogryposis multiplex congenita (AMC) is a group of disorders characterized by congenital joint contractures caused by reduced fetal movements. AMC has an incidence of 1 in 3000 newborns and is genetically heterogeneous. We describe an autosomal recessive form of myogenic AMC in a large consanguineous family. The disease is characterized by bilateral clubfoot, decreased fetal movements, delay in motor milestones, then progressive motor decline after the first decade. Genome-wide linkage analysis revealed a single locus on chromosome 6q25 with Z(max) = 3.55 at theta = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Homozygous A to G nucleotide substitution of the conserved AG splice acceptor site at the junction of intron 136 and exon 137 of the SYNE-1 gene was found in patients. This mutation results in an aberrant retention of intron 136 of SYNE-1 RNA leading to premature stop codons and the lack of the C-terminal transmembrane domain KASH of nesprin-1, the SYNE-1 gene product. Mice lacking the KASH domain of nesprin-1 display a myopathic phenotype similar to that observed in patients. Altogether, these data strongly suggest that the splice site mutation of SYNE-1 gene found in the family is responsible for AMC. Recent reports have shown that mutations of the SYNE-1 gene might be responsible for autosomal recessive adult onset cerebellar ataxia. These data indicate that mutations of nesprin-1 which interacts with lamin A/C may lead to at least two distinct human disease phenotypes, myopathic or neurological, a feature similar to that found in laminopathies.
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Sarcomere mutations in cardiogenesis and ventricular noncompaction.
Trends Cardiovasc. Med.
PUBLISHED: 05-27-2009
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Ventricular noncompaction is a form of cardiomyopathy where increased trabeculation is present frequently affecting the left ventricle and resembling an embryonic state of heart development. Clinically, left ventricular noncompaction may manifest as congestive heart failure, arrhythmias, and/or thromboembolic events. There are multiple genes linked to noncompaction, but recently, sarcomere gene mutations were found in both familial and sporadic cases of noncompaction. The association of noncompaction with sarcomere mutations supports the classification of ventricular noncompaction as cardiomyopathy and raises interesting questions regarding the continuum of hypertrophic cardiomyopathy, dilated cardiomyopathy, and noncompaction. The mutational spectrum of sarcomere genes in these disorders highlights the importance of the MYH7 gene encoding beta-myosin heavy chain and ACTC1 encoding the cardiac actin gene. Intriguingly, these mutations also share a low but definitive incidence of congenital heart malformations including septal defects. These human genetic findings support that normal myocardial and sarcomere function are required for proper compaction and septation and that these mutations also portend a high risk of developing heart failure in later life.
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Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.
J. Clin. Invest.
PUBLISHED: 05-12-2009
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Most single-gene diseases, including muscular dystrophy, display a nonuniform phenotype. Phenotypic variability arises, in part, due to the presence of genetic modifiers that enhance or suppress the disease process. We employed an unbiased mapping approach to search for genes that modify muscular dystrophy in mice. In a genome-wide scan, we identified a single strong locus on chromosome 7 that influenced two pathological features of muscular dystrophy, muscle membrane permeability and muscle fibrosis. Within this genomic interval, an insertion/deletion polymorphism of 36 bp in the coding region of the latent TGF-beta-binding protein 4 gene (Ltbp4) was found. Ltbp4 encodes a latent TGF-beta-binding protein that sequesters TGF-beta and regulates its availability for binding to the TGF-beta receptor. Insertion of 12 amino acids into the proline-rich region of LTBP4 reduced proteolytic cleavage and was associated with reduced TGF-beta signaling, decreased fibrosis, and improved muscle pathology in a mouse model of muscular dystrophy. In contrast, a 12-amino-acid deletion in LTBP4 was associated with increased proteolysis, SMAD signaling, and fibrosis. These data identify Ltbp4 as a target gene to regulate TGF-beta signaling and modify outcomes in muscular dystrophy.
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New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains.
Nucleic Acids Res.
PUBLISHED: 03-24-2009
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Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples.
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NO more muscle fatigue.
J. Clin. Invest.
PUBLISHED: 03-24-2009
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NOS is a key enzyme in the production of NO, a molecule that directly regulates vasorelaxation and blood supply. Diverse forms of muscle disease have been clinically associated with unusual fatigue after exercise. The localization of neuronal NOS (nNOS) at the plasma membrane of muscle has recently been shown to prevent muscle fatigue after exercise. In this issue of the JCI, Lai et al. show that dystrophin--the structural protein missing in individuals with Duchenne muscular dystrophy--anchors nNOS to the sarcolemma through a direct interaction with dystrophin spectrin-like repeats 16 and 17 (see the related article, doi:10.1172/JCI36612). Furthermore, in another recently reported study of mouse models of muscular dystrophy, phosphodiesterase 5A inhibitors were used to treat the downstream ischemia that is associated with nNOS mislocalization. Collectively, these findings significantly advance our understanding of exercise-induced muscle fatigue and its role in muscle disease.
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Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice.
Hum. Mol. Genet.
PUBLISHED: 02-28-2009
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Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprins luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1alpha, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.
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LTBP4 genotype predicts age of ambulatory loss in duchenne muscular dystrophy.
Ann. Neurol.
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OBJECTIVE: Duchenne muscular dystrophy (DMD) displays a clinical range that is not fully explained by the primary DMD mutations. Ltbp4, encoding latent transforming growth factor-? binding protein 4, was previously discovered in a genome-wide scan as a modifier of murine muscular dystrophy. We sought to determine whether LTBP4 genotype influenced DMD severity in a large patient cohort. METHODS: We analyzed nonsynonymous single nucleotide polymorphisms (SNPs) from human LTBP4 in 254 nonambulatory subjects with known DMD mutations. These SNPs, V194I, T787A, T820A, and T1140M, form the VTTT and IAAM LTBP4 haplotypes. RESULTS: Individuals homozygous for the IAAM LTBP4 haplotype remained ambulatory significantly longer than those heterozygous or homozygous for the VTTT haplotype. Glucocorticoid-treated patients who were IAAM homozygotes lost ambulation at 12.5 ± 3.3 years compared to 10.7 ± 2.1 years for treated VTTT heterozygotes or homozygotes. IAAM fibroblasts exposed to transforming growth factor (TGF) ? displayed reduced phospho-SMAD signaling compared to VTTT fibroblasts, consistent with LTBP4 role as a regulator of TGF?. INTERPRETATION: LTBP4 haplotype influences age at loss of ambulation, and should be considered in the management of DMD patients. ANN NEUROL 2013.
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Genetic pathways of vascular calcification.
Trends Cardiovasc. Med.
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Vascular calcification is an independent risk factor for cardiovascular disease. Arterial calcification of the aorta and coronary, carotid, and peripheral arteries becomes more prevalent with age. Genome-wide association studies have identified regions of the genome linked to vascular calcification, and these same regions are linked to myocardial infarction risk. The 9p21 region linked to vascular disease and inflammation also associates with vascular calcification. In addition to these common variants, rare genetic defects can serve as primary triggers of accelerated and premature calcification. Infancy-associated calcific disorders are caused by loss-of-function mutations in ENPP1, an enzyme that produces extracellular pyrophosphate. Adult-onset vascular calcification is linked to mutations in NTE5, another enzyme that regulates extracellular phosphate metabolism. Common conditions that secondarily enhance vascular calcification include atherosclerosis, metabolic dysfunction, diabetes, and impaired renal clearance. Oxidative stress and vascular inflammation, along with biophysical properties, converge with these predisposing factors to promote soft tissue mineralization. Vascular calcification is accompanied by an osteogenic profile, and this osteogenic conversion is seen within the vascular smooth muscle as well as the matrix. Here, we review the genetic causes of medial calcification in the smooth muscle layer, focusing on recent discoveries of gene mutations that regulate extracellular matrix phosphate production and the role of S100 proteins as promoters of vascular calcification.
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The attachment disorders of muscle: failure to carb-load.
J. Clin. Invest.
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Dystroglycan is a prominent cell surface protein that mediates attachment to the extracellular matrix. Although broadly expressed, glycosylated dystroglycan is critically important for muscle cell adherence to its surrounding matrix. A subgroup of muscular dystrophies, which often manifest in infancy, is associated with reduced glycosylation of dystroglycan. In this issue of the JCI, Beedle et al. used conditional gene targeting of Fktn, the gene responsible for Fukuyama congenital muscular dystrophy, to investigate a developmental requirement for glycosylation of dystroglycan.
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S100A12 expression in thoracic aortic aneurysm is associated with increased risk of dissection and perioperative complications.
J. Am. Coll. Cardiol.
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The purpose of this study was to determine the relevance of S100A12 expression to human thoracic aortic aneurysms and type A thoracic aortic aneurysm dissection and to study mechanisms of S100A12-mediated dysfunction of aortic smooth muscle cells.
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Population-based variation in cardiomyopathy genes.
Circ Cardiovasc Genet
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Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes Project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and, therefore, is a rich source to determine both common and rare genetic variants.
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TBX5 drives Scn5a expression to regulate cardiac conduction system function.
J. Clin. Invest.
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Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Nav1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease.
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Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor-? pathway.
Proc. Natl. Acad. Sci. U.S.A.
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The muscular dystrophies are broadly classified as muscle wasting diseases with myofiber dropout due to cellular necrosis, inflammation, alterations in extracellular matrix composition, and fatty cell replacement. These events transpire and progress despite ongoing myofiber regeneration from endogenous satellite cells. The degeneration/regeneration response to muscle injury/disease is modulated by the proinflammatory cytokine transforming growth factor-? (TGF-?), which can also profoundly influence extracellular matrix composition through increased secretion of profibrotic proteins, such as the matricellular protein periostin. Here we show that up-regulation and secretion of periostin is pathological and enhances disease in the ?-sarcoglycan null (Sgcd(-/-)) mouse model of muscular dystrophy (MD). Indeed, MD mice lacking the Postn gene showed dramatic improvement in skeletal muscle structure and function. Mechanistically, Postn gene deletion altered TGF-? signaling so that it now enhanced tissue regeneration with reduced levels of fibrosis. Systemic antagonism of TGF-? with a neutralizing monoclonal antibody mitigated the beneficial effects of Postn deletion in vivo. These data suggest that periostin functions as a disease determinant in MD by promoting/allowing the pathological effects of TGF-?, suggesting that inhibition of periostin could represent a unique treatment approach.
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Interplay between heart and skeletal muscle disease in heart failure: the 2011 George E. Brown Memorial Lecture.
Circ. Res.
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The study of single gene disorders often provides insight for more complex human disease. Mutations in the genes encoding the dystrophin protein complex cause muscular dystrophy and cardiomyopathy by destabilizing the plasma membrane of skeletal myofibers and cardiomyocytes. In these diseases, progressive skeletal muscle degeneration and weakness contribute to cardiac dysfunction. Moreover, the pace and pattern of muscle weakness, along with onset of cardiomyopathy, is highly variable even when associated with the same identical mutation. Using a mouse model of muscular dystrophy and cardiomyopathy, we identified genetic loci that modify muscle pathology and cardiac fibrosis. Distinct genetic modifiers were identified for diaphragm and abdominal musculature, and these genetic intervals differ from those that regulate pathology in the skeletal muscle of the limbs and the heart. One modifier gene was identified and highlights the importance of the transforming growth factor-? pathway in the pathogenesis of muscular dystrophy and cardiomyopathy. We determined that canonical transforming growth factor-? signaling contributes to heart and muscle dysfunction using a Drosophila model. Together, these studies demonstrate the value of using a genetically sensitized model to uncover pathways that regulate heart failure and muscle weakness.
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A model for the ultrastructure of bone based on electron microscopy of ion-milled sections.
PLoS ONE
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The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures ("mineral structures") are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.