[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β) is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5). Antisense oligonucleotides (AONs) were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD.
[Show abstract][Hide abstract] ABSTRACT: Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.
[Show abstract][Hide abstract] ABSTRACT: The importance of adequate level of muscle size and function and physical activity are widely recognized. Myostatin/activin blocking increases skeletal muscle mass, but may decrease muscle oxidative capacity and can thus be hypothesized to affect voluntary physical activity. Soluble activin receptor IIB (sActRIIB-Fc) was produced to block myostatin/activins. Modestly dystrophic mdx mice were injected with sActRIIB-Fc or PBS with or without voluntary wheel running exercise for 7wk. Healthy mice served as controls. Running for 7wk attenuated the sActRIIB-Fc-induced increase in body mass by decreasing fat mass. Running also enhanced/restored the markers of muscle oxidative capacity and autophagy in mdx mice to or above the levels of healthy mice. Voluntary running activity was decreased by sActRIIB-Fc during the first 3-4wk correlating with increased body mass. Homecage physical activity of mice, quantified from the force plate signal, was decreased by sActRIIB-Fc the whole 7wk treatment in sedentary mice. To understand what happens during the first weeks after sActRIIB-Fc administration when mice are less active, healthy mice were injected with sActRIIB-Fc or PBS for 2wk. During the sActRIIB-Fc-induced rapid 2-wk muscle growth period, oxidative capacity and autophagy were reduced, which may possibly explain the decreased running activity. These results show that increased muscle size and decreased markers of oxidative capacity and autophagy during the first weeks of myostatin/activin blocking are associated with decreased voluntary activity levels. Voluntary exercise in dystrophic mice enhances the markers of oxidative capacity and autophagy to or above the levels of healthy mice.
Full-text · Article · May 2013 · AJP Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Loss of muscle mass and function occurs in various diseases. Myostatin blocking can attenuate muscle loss, but downstream signaling is not well known. Therefore, to elucidate associated signaling pathways, we used the soluble activin receptor IIb (sActRIIB-Fc) to block myostatin and activins in mice. Within two weeks, the treatment rapidly increased muscle size as expected, but decreased capillary density per area. sActRIIB-Fc increased muscle protein synthesis 1-2 days after the treatment correlating with enhanced mTORC1 signaling (phosphorylated rpS6 and S6K1, r=0.8). Concurrently, increased REDD1 and eIF2Bε protein contents and phosphorylation of 4EBP1 and AMPK was observed. In contrast, proangiogenic MAPK signaling and VEGF-A protein decreased. Hippo signaling is recently characterized regulator of organ size and an important regulator of myogenesis in vitro. The phosphorylation of YAP (Yes-Associated-Protein), a readout of activated Hippo signaling, increased after short and longer term myostatin and activin blocking and in exercised muscle. Moreover, dystrophic mdx mice had elevated phosphorylated and especially total YAP protein content. These results show that the blocking of myostatin and activins induce rapid skeletal muscle growth. This is associated with increased protein synthesis and mTORC1 signaling, but decreased capillary density and proangiogenic signaling. It is also shown for the first time that Hippo signaling is activated in skeletal muscle after myostatin blocking and exercise and also in dystrophic muscle. This suggests that Hippo signaling may have a role in skeletal muscle in various circumstances.
Full-text · Article · Oct 2012 · AJP Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Abstract Adeno-associated virus (AAV)-U7-mediated skipping of dystrophin-exon-23 restores dystrophin expression and muscle function in the mdx mouse model of Duchenne muscular dystrophy. Soluble activin receptor IIB (sActRIIB-Fc) inhibits signaling of myostatin and homologous molecules and increases muscle mass and function of wild-type and mdx mice. We hypothesized that combined treatment with AAV-U7 and sActRIIB-Fc may synergistically improve mdx muscle function. Bioactivity of sActRIIB-Fc on skeletal muscle was first demonstrated in wild-type mice. In mdx mice we show that AAV-U7-mediated dystrophin restoration improved specific muscle force and resistance to eccentric contractions when applied alone. Treatment of mdx mice with sActRIIB-Fc increased body weight, muscle mass and myofiber size, but had little effect on muscle function. Combined treatment stimulated muscle growth comparable to the effect of sActRIIB-Fc alone and dystrophin rescue was similar to AAV-U7 alone. Moreover, combined treatment improved maximal tetanic force and the resistance to eccentric contraction to similar extent as AAV-U7 alone. In conclusion, combination of dystrophin exon skipping with sActRIIB-Fc brings together benefits of each treatment; however, we failed to evidence a clear synergistic effect on mdx muscle function.
Full-text · Article · Aug 2012 · Human gene therapy
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) are important extracellular cytokines that play critical roles in embryogenesis and tissue homeostasis. BMPs signal via transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad effector proteins. BMP signaling is precisely regulated and perturbation of BMP signaling is connected to multiple diseases, including musculoskeletal diseases. In this review, we will summarize the recent progress in elucidation of BMP signal transduction, how overactive BMP signaling is involved in the pathogenesis of heterotopic ossification and Duchenne muscular dystrophy, and discuss possible therapeutic strategies for treatment of these diseases.
Full-text · Article · Jul 2012 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: Since the discovery of the myostatin/ActRIIB signaling pathway 15 years ago, numerous strategies were developed to block its inhibitory function during skeletal muscle growth. Accumulating evidence demonstrates that abrogation of myostatin/ActRIIB signaling ameliorates pathology and function of dystrophic muscle in animal models for Duchenne muscular dystrophy (DMD). Therapeutic trials in healthy man and muscular dystrophy patients suggest feasibility of blockade strategies for potential clinical use. However, many key questions on the effect of myostatin/ActRIIB blockade remain unresolved; such as the underlying molecular mechanism that triggers muscle growth, the effect on muscle regeneration and adult muscle stem cell regulation and whether it causes long term metabolic alterations. Current therapeutic strategies aim to systemically abrogate myostatin/ActRIIB signaling. Although this ensures widespread effect on musculature, it also interferes with ActRIIB signaling in other tissues than skeletal muscle, thereby risking adverse effects. This review discusses current knowledge on myostatin/ActRIIB signaling and its potential value as a therapeutic target for DMD.
No preview · Article · Apr 2012 · Current Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: A key step in heart development is the coor-dinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that elec-trically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coor-dination of these important processes in heart development.
Full-text · Article · Apr 2012 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: Treatment of disorders of the sinus node or the atrioventricular node requires insights into the molecular mechanisms of development and homoeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogramme terminally differentiated working cardiomyocytes into pacemaker cells.
TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice using tamoxifen. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na(+) channel (Na(V)1.5; I(Na)), and inwardly rectifying K(+) ion channels (K(ir) genes; I(K1)). Concordantly, we observed conduction slowing in these hearts and reductions in I(Na) and I(K1) in cardiomyocytes isolated from these hearts. The reduction in I(K1) resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current I(f) was observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes.
TBX3 reprogrammes terminally differentiated working cardiomyocytes and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce I(K1) density to enable diastolic depolarization are promising TBX3 characteristics that may facilitate biological pacemaker formation strategies.
No preview · Article · Mar 2012 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: The TGF-β protein family consists of secreted multifunctional cytokines that control diverse processes, such as cell growth and differentiation. Aberrant expression and downstream signaling of these growth factors have been associated with multiple diseases, including muscle wasting disorders, such as Duchenne muscular dystrophy. In this review we discuss recent advances in understanding the role of TGF-β family members during normal skeletal muscle biology/regeneration and their role in muscle pathology, with a special focus on Duchenne muscular dystrophy. In addition, we will highlight progress in the development of potential therapeutics for Duchenne muscular dystrophy based on intervention of TGF-β signaling.
No preview · Article · Mar 2012 · Future Neurology
[Show abstract][Hide abstract] ABSTRACT: The genetic defect of mdx mice resembles that of Duchenne muscular dystrophy, although their functional performance and life expectancy is nearly normal. By contrast, mice lacking utrophin and dystrophin (mdx/utrn -/-) are severely affected and die prematurely. Mice with one utrophin allele (mdx/utrn +/-) are more severely affected than mdx mice, but outlive mdx/utrn -/- mice. We subjected mdx/utrn +/+, +/-, -/- and wild type males to a 12week functional test regime of four different functional tests. Mdx/utrn +/+ and +/- mice completed the regime, while mdx/utrn -/- mice died prematurely. Mdx/utrn +/- mice performed significantly worse compared to mdx/utrn +/+ mice in functional tests. Creatine kinase levels, percentage of fibrotic/necrotic tissue, morphology of neuromuscular synapses and expression of biomarker genes were comparable, whereas mdx/utrn +/- and -/- mice had increased levels of regenerating fibers. This makes mdx/utrn +/- mice valuable for testing the benefit of potential therapies on muscle function parameters.
Full-text · Article · Jan 2012 · Neuromuscular Disorders
[Show abstract][Hide abstract] ABSTRACT: The transforming growth factor (TGF)-β family member myostatin is an important regulator of myoblast, adipocyte, and fibroblast growth and differentiation, but the signaling mechanisms remain to be established. We therefore determined the contribution of myostatin type I receptors activin receptor-like kinase-4 (ALK4) and -5 (ALK5) and different coreceptors in C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, and 3T3-L1 fibroblasts, as well as in primary myoblast and fibroblasts. We performed siRNA-mediated knockdown of each receptor and measured signaling activity using Smad3-dependent luciferase and Smad2 phosphorylation assays with nontargeting siRNA as control. We find that myostatin utilizes ALK4 in myoblasts, whereas it has a preference for ALK5 in nonmyogenic cells. Notably, our results show that coreceptor Cripto is expressed in myoblasts but not in the nonmyogenic cells and that it regulates myostatin activity. More specifically, myostatin requires Cripto in myoblasts, whereas Cripto represses activin activity and TGF-β signaling is Cripto independent. Cripto-mediated myostatin signaling is dependent on both epidermal growth factor (EGF)-like and Cripto-FRL1-cryptic (CFC) domains, whereas activin signaling is solely conferred by the CFC domain. Furthermore, Cripto down-regulation enhances myoblast differentiation, showing its importance in myostatin signaling. Together, our results identify a molecular mechanism that explains the cell-type specific aspects of signaling by myostatin and other TGF-β family members.
[Show abstract][Hide abstract] ABSTRACT: A key step in heart development is the coordinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that electrically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coordination of these important processes in heart development.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-011-0884-2) contains supplementary material, which is available to authorized users.
Full-text · Article · Dec 2011 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β) family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD) is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients.
We targeted myostatin exon 2 using antisense oligonucleotides (AON) in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections.
Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes.
We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.
Full-text · Article · Apr 2011 · BMC Medical Genomics
[Show abstract][Hide abstract] ABSTRACT: This chapter focuses on the cellular origin of the conduction system components and the molecular genetic mechanisms that may control their phenotype and position within the developing heart. It discusses the connection between heart cell precursor pools, which in a temporal pattern form the heart, and the genesis of the conduction system components. The distinct components of the cardiac conduction system of the heart are essentially myocardial. They are innervated by cardiac ganglia largely derived from neural crest. In addition, a large fraction of cells in the mature conduction system is noncardiac, and insulating layers of fibrous tissue are found around conduction system components, such as the SAN and AV bundle. These noncardiac cell types are derived from the epicardium (fibroblasts), endocardium, neural crest (neural innervations), and other sources, although their origins have not been defined in detail. Although these nerves and fibrous tissues are important, or even a prerequisite, for conduction system formation and function, the cardiomyocytes are essential for impulse generation and propagation. Furthermore, in the embryo the functional myocardial conduction system is not yet innervated and interstitial fibroblast and fibrous tissues in association with the conduction system are sparse or absent. Studies of the development of the conduction system components strongly suggest that they originate from myocardial precursors, which in turn are derived from mesoderm and pericardial wall mesenchyme.
[Show abstract][Hide abstract] ABSTRACT: Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.
Full-text · Article · Oct 2010 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: The complex four-chambered heart is an outcome of regionalization, patterning and differentiation
during embryonic development. In the developing heart, chamber myocardium of atrium and ventricles
are separated and bordered by non-chamber myocardium of the atrioventricular canal (avc) and
outflow tract (oft). The avc myocardium is required for aligning the atrial and ventricular chambers and
for mesenchymal cushion formation. Cushion tissues are subsequently deployed in the formation of
valves and septa. Work from the last decade has shown that T-box proteins, which are expressed in
different compartments of the developing heart in exclusive and sometimes overlapping fashion, are
downstream effectors of several signaling morphogens, particularly BMPs, and play crucial role in
cardiac regionalization. Tbx2 is expressed in and required for the formation of the avc (Harrelson 2004;
Aanhaanen 2009). However, Tbx2 mutants showed variable phenotypes with incomplete penetrance.
Since the closely related paralog Tbx3 is co-expressed with Tbx2 in the avc, we generated and
analyzed Tbx2/Tbx3 double mutants in order to eliminate the effect of redundancy. Our analysis
indeed suggested that Tbx2 in combination with Tbx3 is crucial for avc establishment by suppressing
chamber myocardial gene programs in this particular domain of the heart. Simultaneously, we also
observed the insufficiency of double mutants to maintain Bmp2 expression and to induce cardiac jelly,
the very first step of cushion formation. By gain of function studies we could show that Tbx2 and Tbx3
are individually sufficient for inducing cushion formation by activating Bmp2 and thereby acting in a
[Show abstract][Hide abstract] ABSTRACT: Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Nav1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Nav1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Nav1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Nav1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-009-0012-8) contains supplementary material, which is available to authorized users.
Full-text · Article · Apr 2009 · Archiv für Kreislaufforschung
[Show abstract][Hide abstract] ABSTRACT: Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data
are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence
baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values
and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because
of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of
the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing
the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible
PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points
in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of
these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.
Full-text · Article · Mar 2009 · Nucleic Acids Research