The mouse dystrophin muscle enhancer-1 imparts skeletal muscle, but not cardiac muscle, expression onto the dystrophin Purkinje promoter in transgenic mice

A subset of patients harboring mutations in the dystrophin gene suffer from X-linked dilated cardiomyopathy (XLCM), a familial heart disease that is not accompanied by any clinical signs of skeletal muscle myopathy. As the muscle (M) isoform of dystrophin is not expressed in these patients, the absence of skeletal muscle symptoms has been attributed to expression of the brain (B) and cerebellar Purkinje (CP) isoforms of dystrophin in skeletal, but not cardiac, muscles of XLCM patients. The compensatory mechanism of dystrophin B and CP promoter upregulation is not known but it has been suggested that the dystrophin muscle enhancer from intron 1, DME-1, may be important in this activity. Previous studies have shown that the presence of the DME-1 is essential for a significant increase in dystrophin B and CP promoter activity in skeletal muscle cells in culture. Here, we demonstrate that the mouse dystrophin CP promoter drives expression of a lacZ reporter gene specifically to the cerebellar Purkinje cell layer but not to skeletal or cardiac muscle of transgenic mice. However, if the mouse counterpart of DME-1 is present in the transgene construct, the dystrophin CP promoter is now activated in skeletal muscle, but not in cardiac muscle. Our findings provide in vivo evidence for the importance of the dystrophin muscle enhancer sequences in activating the dystrophin CP promoter in skeletal muscle. Furthermore, they provide support for the model in which muscle enhancers, like DME-1, activate the dystrophin B and CP promoters in skeletal muscle, but not in cardiac muscle, of XLCM patients.     

A point mutation in the 5' splice site of the dystrophin gene first intron responsible for X-linked dilated cardiomyopathy

X-linked dilated cardiomyopathy (XLDC) is a familial heart disease presenting in young males as a rapidly progressive congestive heart failure, without clinical signs of skeletal myopathy. This condition has recently been linked to the dystrophin gene in some families and deletions encompassing the genomic region coding for the first muscle exon have been detected. In order to identify the defect responsible for this disease at the molecular level and to understand the reasons for the selective heart involvement, a family with a severe form of XLDC was studied. In the affected members, no deletions of the dystrophin gene were observed. Analysis of the muscle promoter, first exon and intron regions revealed the presence of a single point mutation at the first exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the first intron. This mutation introduced a new restriction site for MseI, which cosegregates with the disease in the analyzed family. Expression of the major dystrophin mRNA isoforms (from the muscle-, brain- and Purkinje cell-promoters) was completely abolished in the myocardium, while the brain- and Purkinje cell- (but not the muscle-) isoforms were detectable in the skeletal muscle. Immunocytochemical studies with anti- dystrophin antibodies showed that the protein was reduced in quantity but normally distributed in the skeletal muscle, while it was undetectable in the cardiac muscle. These findings indicate that expression of the muscle dystrophin isoform is critical for myocardial function and suggest that selective heart involvement in dystrophin- linked dilated cardiomyopathy is related to the absence, in the heart, of a compensatory expression of dystrophin from alternative promoters.      

Mouse dystrophin enhancer preferentially targets lacZ expression in skeletal and cardiac muscle.

Duchenne muscular dystrophy is a muscle wasting disease that results from a dystrophin deficiency in skeletal and cardiac muscle. Studies concerning the regulatory elements that govern dystrophin gene expression in skeletal and/or cardiac muscle in both mouse and human have identified a promoter and an enhancer located in intron 1. In transgenic mice, the muscle promoter alone targets the expression of a lacZ reporter gene only to the right ventricle of the heart, suggesting the need for other regulatory elements to target skeletal muscle and the rest of the heart. Here we report that the mouse dystrophin enhancer from intron 1 can target the expression of a lacZ reporter gene in skeletal muscle as well as in other heart compartments of transgenic mice. Our results also suggest that sequences surrounding the mouse dystrophin enhancer may affect its function throughout mouse development.     

Mutations in the dystrophin gene are associated with sporadic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is the major indication for heart transplantation. Approximately 30% of all DCM is thought to be inherited, while 70% is sporadic. Mutations in the dystrophin gene have been associated with the uncommon X-linked form of DCM. We hypothesized that missense mutations and other less severe mutations of the dystrophin gene might predispose to the common form of sporadic DCM. To test this hypothesis, 22kb of genomic dystrophin DNA was scanned with DOVAM-S in each of the 22 patients with sporadic DCM, including all 79 coding sequences and splice junctions, as well as six alternative exon 1 dystrophin isoforms (484kb, total). Three putative new mutations (IVS5+1 G>T, K18N, and F3228L) and seven polymorphisms were identified. The splice site mutation IVS5+1 is predicted to cause skipping of exon 5, which is within a region containing an actin binding site. The missense mutations occur at amino acids that display substantial evolutionary conservation. Screening of 236 control individuals failed to identify these three mutations. The three patients with putative mutations had CK-MM (creatine kinase, skeletal muscle) levels greater than 250 units while the 14 patients without mutations for which CK-MM were available had values ranging from 20 to 200. The first comprehensive mutation scanning of the exons and splice junctions of the dystrophin gene in patients with sporadic DCM presents the evidence that point mutations are associated with sporadic DCM without clinical evidence of skeletal myopathy. It may be prudent to measure CK-MM in all patients with dilated cardiomyopathy to identify candidates at high risk for dystrophin mutations.     

Long-term rescue of dystrophin expression and improvement in muscle pathology and function in dystrophic mdx mice by peptide-conjugated morpholino

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2'-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD(50) of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.     

Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters.

To test tissue specificity of zebrafish gene promoters in a heterologous fish species, two transgenic medaka lines under two zebrafish promoters were generated. Under the zebrafish skeletal muscle-specific mylz2 promoter, transgenic medaka expressed green fluorescent protein (GFP) exclusively in skeletal muscles, mimicking the endogenous medaka mylz2 mRNA expression and also identical to GFP expression in mylz2:gfp transgenic zebrafish. A madaka mylz2 promoter was also capable of directing skeletal muscle-specific GFP expression in transient transgenic zebrafish embryos. In the krt8:rfp transgenic medaka line with the zebrafish epithelial krt8 promoter, red fluorescent protein was specifically expressed in the skin epithelia as well as the epithelial lining cells of the anterior digestive tract, which was also identical to GFP expression in krt8:gfp transgenic zebrafish. Therefore, the two zebrafish promoters faithfully function in a heterologous fish species, and it is likely that the mechanisms of tissue-specific expression are largely conserved among fish species.     

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

Aim: The dystrophin–glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow‐type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast‐type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue‐resistant characteristics of skeletal muscle. Methods: We measured the expression of dystrophin and β1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. Results: The expression of dystrophin and β1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast‐type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Conclusion: Our results support the idea that DGC and FAC are among the factors that explain the fatigue‐resistant property not only of slow‐type but also of fast‐type skeletal muscles.     

Developmental regulation of MURF E3 ubiquitin ligases in skeletal muscle

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as E3 ubiquitin ligases in ubiquitin-mediated muscle protein turnover. Despite the well-characterised role of MURF1 in skeletal muscle atrophy, the dynamics of MURF isogene expression in the development and early postnatal adaptation of skeletal muscle is unknown. Here, we show that MURF2 is the isogene most highly expressed in embryonic skeletal muscle at E15.5, with the 50 kDa A isoform predominantly expressed. MURF1 and MURF3 are upregulated only postnatally. Knockdown of MURF2 p50A by isoform-specific siRNA results in delayed myogenic differentiation and myotube formation in vitro, with perturbation of the stable, glutamylated microtubule population. This underscores that MURF2 plays an important role in the earliest stages of skeletal muscle differentiation and myofibrillogenesis. During further development, there is a shift towards the 60 kDa A isoform, which dominates postnatally. Analysis of the fibre-type expression shows that MURF2 A isoforms are predominantly slow-fibre associated, whilst MURF1 is largely excluded from these fibres, and MURF3 is ubiquitously distributed in both type I and II fibres.     

Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.