Michele Hadhazy

Dominant negative myostatin produces hypertrophy without hyperplasia in muscle

Myostatin, a TGF‐β family member, is a negative regulator of muscle growth. Here, we generated transgenic mice that expressed myostatin mutated at its cleavage site under the control of a muscle specific promoter creating a dominant negative myostatin. These mice exhibited a significant (20–35%) increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. We also evaluated the role of myostatin in muscle degenerative states, such as muscular dystrophy, and found significant downregulation of myostatin. Thus, further inhibition of myostatin may permit increased muscle growth in muscle degenerative disorders.

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.

Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice

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.

Latent TGF-β–binding protein 4 modifies muscular dystrophy in mice

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.

Spontaneous Coronary Vasospasm in KATP Mutant Mice Arises From a Smooth Muscle–Extrinsic Process

In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle–specific SM22&agr; promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle–extrinsic process.

Smooth muscle cell-extrinsic vascular spasm arises from cardiomyocyte degeneration in sarcoglycan-deficient cardiomyopathy

Vascular spasm is a poorly understood but critical biomedical process because it can acutely reduce blood supply and tissue oxygenation. Cardiomyopathy in mice lacking gamma-sarcoglycan or delta-sarcoglycan is characterized by focal damage. In the heart, sarcoglycan gene mutations produce regional defects in membrane permeability and focal degeneration, and it was hypothesized that vascular spasm was responsible for this focal necrosis. Supporting this notion, vascular spasm was noted in coronary arteries, and disruption of the sarcoglycan complex was observed in vascular smooth muscle providing a molecular mechanism for spasm. Using a transgene rescue strategy in the background of sarcoglycan-null mice, we replaced cardiomyocyte sarcoglycan expression. Cardiomyocyte-specific sarcoglycan expression was sufficient to correct cardiac focal degeneration. Intriguingly, successful restoration of the cardiomyocyte sarcoglycan complex also eliminated coronary artery vascular spasm, while restoration of smooth muscle sarcoglycan in the background of sarcoglycan-null alleles did not. This mechanism, whereby tissue damage leads to vascular spasm, can be partially corrected by NO synthase inhibitors. Therefore, we propose that cytokine release from damaged cardiomyocytes can feed back to produce vascular spasm. Moreover, vascular spasm feeds forward to produce additional cardiac damage.

Genetic background influences muscular dystrophy

Mutations in the genes encoding dystrophin and its associated proteins, the sarcoglycans, lead to muscular dystrophy in humans and in mouse models. In the presence of identical gene mutations, the muscular dystrophy phenotype can be highly variable. Using a mouse model of limb girdle muscular dystrophy engineered with a null allele of gamma-sarcoglycan, we bred the identical gamma-sarcoglycan mutation into four different genetic backgrounds. We found that the gamma-sarcoglycan mutation is least severe in the129SV/J (129) strain and most severe on the DBA 2J JAX (DBA) strain using quantitative measures of Evans blue dye uptake, as a marker of membrane permeability defects, and hydroxyproline content, as a marker of fibrosis. In addition we show that the DBA mice are most severely affected regardless of gender and age. The enhanced phenotype observed in the DBA strain was not caused by exercise as the DBA mice scored the lowest in a voluntary activity test. The milder phenotype seen in the 129SV/J and C57B6/J strains suggests that these backgrounds contain modifier loci that partially suppress the muscular dystrophy phenotype. Identification of these modifier genes and the associated pathways may lead to novel therapeutic strategies.

Cardiomyopathy is independent of skeletal muscle disease in muscular dystrophy

Dystrophin and its associated proteins, the sarcoglycans, are normally expressed in heart and skeletal muscle. Mutations that alter the expression of these membrane‐associated proteins lead to muscular dystrophy (MD) and cardiomyopathy in humans. Because of the timing and nature of the accompanying cardiomyopathy, it has been suggested that cardiomyopathy develops as a secondary consequence of skeletal muscle dysfunction in the muscular dystrophies. To determine whether skeletal muscle dystrophy contributes to the development of sarcoglycan‐mediated cardiomyopathy, we used mice lacking γ‐sarcoglycan and inserted a transgene that “rescued” γ‐sarcoglycan expression only in skeletal muscle. γ‐Sarcoglycan was expressed in skeletal muscle under the control of the skeletal muscle‐specific myosin light chain 1/3 promoter. γ‐Sarcoglycannull mice expressing this transgene fully restore γ‐sarcoglycan expression. Furthermore, the transgene‐rescued mice lack the focal necrosis and membrane permeability defects that are a hallmark of MD. Despite correction of the skeletal muscle disease, focal degeneration and membrane permeability abnormalities persisted in cardiac muscle, and notably persisted in the right ventricle. Therefore, heart and skeletal muscle defects are independent processes in sarcoglycan‐mediated muscular dystrophies and, as such, therapy should target both skeletal and cardiac muscle correction to prevent sudden death due to cardiomyopathy in the muscular dystrophies.

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

The superhealing MRL background improves muscular dystrophy

BackgroundMice from the MRL or “superhealing” strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis.MethodsMRL/MpJ mice were bred with Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains.ResultsIntroduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis.ConclusionsThese data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease.