A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I.

Myotonic dystrophy Type 1 (DM1) is a rare genetic disease caused by the expansion of CTG trinucleotide repeats ((CTG)exp) in the 3' untranslated region of the DMPK gene. The repeat transcripts sequester the RNA binding protein Muscleblind-like protein 1 (MBNL1) and hamper its normal function in pre-mRNA splicing. Overexpressing exogenous MBNL1 in the DM1 mouse model has been shown to rescue the splicing defects and reverse myotonia. Although a viable therapeutic strategy, pharmacological modulators of MBNL1 expression have not been identified. Here, we engineered a ZsGreen tag into the endogenous MBNL1 locus in HeLa cells and established a flow cytometry-based screening system to identify compounds that increase MBNL1 level. The initial screen of small molecule compound libraries identified more than thirty hits that increased MBNL1 expression greater than double the baseline levels. Further characterization of two hits revealed that the small molecule HDAC inhibitors, ISOX and vorinostat, increased MBNL1 expression in DM1 patient-derived fibroblasts and partially rescued the splicing defect caused by (CUG)exp repeats in these cells. These findings demonstrate the feasibility of this flow-based cytometry screen to identify both small molecule compounds and druggable targets for MBNL1 upregulation.

Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling.

Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.

N-terminal titin fragment: a non-invasive, pharmacodynamic biomarker for microdystrophin efficacy.

BACKGROUND: Multiple clinical trials to assess the efficacy of AAV-directed gene transfer in participants with Duchenne muscular dystrophy (DMD) are ongoing. The success of these trials currently relies on standard functional outcome measures that may exhibit variability within and between participants, rendering their use as sole measures of drug efficacy challenging. Given this, supportive objective biomarkers may be useful in enhancing observed clinical results. Creatine kinase (CK) is traditionally used as a diagnostic biomarker of DMD, but its potential as a robust pharmacodynamic (PD) biomarker is difficult due to the wide variability seen within the same participant over time. Thus, there is a need for the discovery and validation of novel PD biomarkers to further support and bolster traditional outcome measures of efficacy in DMD. METHOD: Potential PD biomarkers in DMD participant urine were examined using a proteomic approach on the Somalogic platform. Findings were confirmed in both mdx mice and Golden Retriever muscular dystrophy (GRMD) dog plasma samples. RESULTS: Changes in the N-terminal fragment of titin, a well-known, previously characterized biomarker of DMD, were correlated with the expression of microdystrophin protein in mice, dogs, and humans. Further, titin levels were sensitive to lower levels of expressed microdystrophin when compared to CK. CONCLUSION: The measurement of objective PD biomarkers such as titin may provide additional confidence in the assessment of the mechanism of action and efficacy in gene therapy clinical trials of DMD. TRIAL REGISTRATION: ClinicalTrials.gov NCT03368742.

Clinical potential of microdystrophin as a surrogate endpoint.

Accelerated approval based on a likely surrogate endpoint can be life-changing for patients suffering from a rare progressive disease with unmet medical need, as it substantially hastens access to potentially lifesaving therapies. In one such example, antisense morpholinos were approved to treat Duchenne muscular dystrophy (DMD) based on measurement of shortened dystrophin in skeletal muscle biopsies as a surrogate biomarker. New, promising therapeutics for DMD include AAV gene therapy to restore another form of dystrophin termed mini- or microdystrophin. AAV-microdystrophins are currently in clinical trials but have yet to be accepted by regulatory agencies as reasonably likely surrogate endpoints. To evaluate microdystrophin expression as a reasonably likely surrogate endpoint for DMD, this review highlights dystrophin biology in the context of functional and clinical benefit to support the argument that microdystrophin proteins have a high probability of providing clinical benefit based on their rational design. Unlike exon-skipping based strategies, the approach of rational design allows for functional capabilities (i.e. quality) of the protein to be maximized with every patient receiving the same optimized microdystrophin. Therefore, the presence of rationally designed microdystrophin in a muscle biopsy is likely to predict clinical benefit and is consequently a strong candidate for a surrogate endpoint analysis to support accelerated approval.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

The structure of the myosin VI motor reveals the mechanism of directionality reversal.

Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.

Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

A structural state of the myosin V motor without bound nucleotide.

The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 A structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

Membrane recruitment of nNOSµ in microdystrophin gene transfer to enhance durability.

Several gene transfer clinical trials are currently ongoing with the common aim of delivering a shortened version of dystrophin, termed a microdystrophin, for the treatment of Duchenne muscular dystrophy (DMD). However, one of the main differences between these trials is the microdystrophin protein produced following treatment. Each gene transfer product is based on different selections of dystrophin domain combinations to assemble microdystrophin transgenes that maintain functional dystrophin domains and fit within the packaging limits of an adeno-associated virus (AAV) vector. While domains involved in mechanical function, such as the actin-binding domain and ß-dystroglycan binding domain, have been identified for many years and included in microdystrophin constructs, more recently the neuronal nitric oxide synthase (nNOS) domain has also been identified due to its role in enhancing nNOS membrane localization. As nNOS membrane localization has been established as an important requirement for prevention of functional ischemia in skeletal muscle, inclusion of the nNOS domain into a microdystrophin construct represents an important consideration. The aim of this mini review is to highlight what is currently known about the nNOS domain of dystrophin and to describe potential implications of this domain in a microdystrophin gene transfer clinical trial.

Transcriptional profiling of testosterone-regulated genes in the skeletal muscle of human immunodeficiency virus-infected men experiencing weight loss.

CONTEXT: HIV-associated wasting and weight loss remain clinically significant concerns even in the era of potent antiretroviral therapy. Although androgen treatment increases muscle mass, the cell-intrinsic mechanisms engaged remain poorly understood. OBJECTIVE: This study was an unbiased approach to identify expression profiles associated with testosterone treatment using genome-wide microarray analysis of skeletal muscle biopsies. DESIGN, SETTING, AND PARTICIPANTS: Forty-four HIV-positive men with weight loss were randomized to receive either 300 mg testosterone enanthate or placebo injections im weekly for 16 wk. Muscle biopsies were obtained at baseline and on treatment d 14. A subset of specimens was chosen for microarray analysis, with changes in selected genes confirmed by real-time PCR, Western blot analysis, and in vitro culture of muscle precursor cells. RESULTS: Significantly greater gains in body mass (+2.05 and -1.07 kg, respectively; P = 0.003) and lean body mass by dual-energy x-ray absorptiometry (2.93 vs. 0.35 kg, respectively; P = 0.003) were observed in subjects treated with testosterone compared with placebo. Microarray analysis revealed up-regulation in genes involved in myogenesis and muscle protein synthesis, immune regulation, metabolic pathways, and chromatin remodeling. Representative genes were confirmed by real-time PCR and protein expression studies. In an independent analysis, gene networks that differentiate healthy young men from older men with sarcopenia had substantial overlap with those activated by testosterone treatment. CONCLUSIONS: These data provide new insights into the mechanisms of androgen action and have implications for both development of muscle biomarkers and anabolic therapies for wasting and sarcopenia.

Structure-Based Approach To Identify 5-[4-Hydroxyphenyl]pyrrole-2-carbonitrile Derivatives as Potent and Tissue Selective Androgen Receptor Modulators.

In an effort to find new and safer treatments for osteoporosis and frailty, we describe a novel series of selective androgen receptor modulators (SARMs). Using a structure-based approach, we identified compound 7, a potent AR (ARE EC50 = 0.34 nM) and selective (N/C interaction EC50 = 1206 nM) modulator. In vivo data, an AR LBD X-ray structure of 7, and further insights from modeling studies of ligand receptor interactions are also presented.

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

BACKGROUND: Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. METHODS: To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. RESULTS: Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. CONCLUSIONS: Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.

Attenuation of skeletal muscle atrophy via protease inhibition.

Skeletal muscle atrophy in response to a number of muscle wasting conditions, including disuse, involves the induction of increased protein breakdown, decreased protein synthesis, and likely a variable component of apoptosis. The increased activation of specific proteases in the atrophy process presents a number of potential therapeutic targets to reduce muscle atrophy via protease inhibition. In this study, mice were provided with food supplemented with the Bowman-Birk inhibitor (BBI), a serine protease inhibitor known to reduce the proteolytic activity of a number of proteases, such as chymotrypsin, trypsin, elastase, cathepsin G, and chymase. Mice fed the BBI diet were suspended for 3-14 days, and the muscle mass and function were then compared with those of the suspended mice on a normal diet. The results indicate that dietary supplementation with BBI significantly attenuates the normal loss of muscle mass and strength following unloading. Furthermore, the data reveal the existence of yet uncharacterized serine proteases that are important contributors to the evolution of disuse atrophy, since BBI inhibited serine protease activity that was elevated following hindlimb unloading and also slowed the loss of muscle fiber size. These results demonstrate that targeted reduction of protein degradation can limit the severity of muscle mass loss following hindlimb unloading. Thus BBI is a candidate therapeutic agent to minimize skeletal muscle atrophy and loss of strength associated with disuse, cachexia, sepsis, weightlessness, or the combination of age and inactivity.

1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)indoline-4-carbonitrile derivatives as potent and tissue selective androgen receptor modulators.

We present a novel series of selective androgen receptor modulators (SARMs) which shows excellent biological activity and physical properties. 1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)-indoline-4-carbonitriles showed potent binding to the androgen receptor (AR) and activated AR-mediated transcription in vitro. Representative compounds demonstrated diminished activity in promoting the intramolecular interaction between the AR carboxyl (C) and amino (N) termini. This N/C-termini interaction is a biomarker assay for the undesired androgenic responses in vivo. In orchidectomized rats, daily administration of a lead compound from this series showed anabolic activity by increasing levator ani muscle weight. Importantly, minimal androgenic effects (increased tissue weights) were observed in the prostate and seminal vesicles, along with minimal repression of circulating luteinizing hormone (LH) levels and no change in the lipid and triglyceride levels. This lead compound completed a two week rat toxicology study, and was well tolerated at doses up to 100 mg/kg/day, the highest dose tested, for 14 consecutive days.

Thin filament activation and unloaded shortening velocity of rabbit skinned muscle fibres.

The unloaded shortening velocity of skinned rabbit psoas muscle fibres is sensitive to [Ca2+]. To determine whether Ca2+ affects the unloaded shortening velocity via regulation of crossbridge kinetics or crossbridge number, the shortening velocity was measured following changes in either [Ca2+] or the number of active thin filament regulatory units. The native troponin C (TnC) was extracted and replaced with either cardiac TnC (cTnC) or a mixture of cTnC and an inactive mutant cardiac TnC (CBMII TnC). The unloaded shortening velocity of the cTnC-replaced fibres was determined at various values of [Ca2+] and compared with different cTnC:CBMII TnC ratios at a saturating [Ca2+]. If Ca2+ regulates the unloaded shortening velocity via kinetic modulation, differences in the velocity-tension relationship between the cTnC fibres and the cTnC:CBMII TnC fibres would be apparent. Alternatively, Ca2+ control of the number of active crossbridges would yield similar velocity-tension relationships when comparing the cTnC and cTnC:CBMII TnC fibres. The results show a decline in the unloaded shortening velocity that is determined by the relative tension, defined as the level of thin filament activation, rather than the [Ca2+]. Furthermore, at lower levels of relative tension, the reduction in unloaded shortening is not the result of changes in any cooperative effects of myosin on Ca2+ binding to the thin filament. Rather, it may be related to a decrease in crossbridge-induced activation of the thin filament at the level of the individual regulatory unit. In summary, the results suggest that Ca2+ regulates the unloaded shortening velocity in skinned fibres by reducing the number of crossbridges able to productively bind to the thin filament without affecting any inherent property of the myosin.

Calcium functionally uncouples the heads of myosin VI.

This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.