Biology subjects

Hammers, D. W.

Publications and source records attributed to Hammers, D. W..

7 recordsLinked to original sources

bioRxiv2025

Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats. MethodsCRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses. ResultsMDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship and a muscle fiber-type shift. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition. ConclusionsMDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.

physiology↗

bioRxiv2025

Dual S100A1 and ARC gene therapy as a treatment for DMD cardiomyopathy

Duchenne muscular dystrophy (DMD) is a lethal pediatric striated muscle disease caused by loss of dystrophin for which there is no cure. Cardiomyopathy is the leading cause of death amongst individuals with DMD, and effective therapeutics to treat DMD cardiomyopathy are a major unmet clinical need. This work investigated adeno-associated viral (AAV) gene therapy approaches to treat DMD cardiomyopathy by overexpression of the calcium binding proteins S100A1 and apoptosis repressor with caspase recruitment domains (ARC). Using the severe D2.mdx mouse model of DMD, we identified that S100A1 gene therapy improves the diastolic dysfunction associated with DMD cardiomyopathy, whereas ARC gene therapy prolongs survival. The combination of both S100A1 and ARC in a single bicistronic vector improves the long-term cardiac outcome of D2.mdx mice, development of heart failure caused by micro-dystrophin expression, and exhibits safety via intracoronary delivery in a canine model of DMD. Furthermore, S100A1-ARC gene therapy provides functional benefits when expressed in D2.mdx skeletal muscle. Together, these findings indicate that S100A1-ARC gene therapy represents an effective treatment for DMD cardiomyopathy and may be effective in treating other forms of cardiomyopathy and muscle pathologies. SIGNIFICANCE STATEMENTCardiomyopathy is the leading cause of death amongst individuals with Duchenne muscular dystrophy (DMD). Effective therapeutics to treat DMD cardiomyopathy represent a major unmet clinical need. This work identifies the dual gene therapy approach of S100A1 and ARC as an effective treatment that improves long-term cardiac function and life-expectancy in severe mouse model of DMD. Intracoronary delivery of this AAV-based gene therapy also exhibits safety and evidence of efficacy in dystrophic canines. Furthermore, functional benefits in skeletal muscle are also incurred via S100A1-ARC expression in striated muscle. These findings indicate that S100A1-ARC therapy is an effective treatment for DMD cardiomyopathy whose benefits may be applicable for other forms of cardiac and muscle disease.

molecular biology↗

bioRxiv2022

Potential limitations of micro-dystrophin gene therapy for Duchenne muscular dystrophy

Adeno-associated viruses (AAVs) expressing versions of truncated dystrophin (micro-dystrophins) are being delivered at high doses to patients with Duchenne muscular dystrophy (DMD) in clinical trials. We examined this strategy with two different micro-dystrophins, similar to those currently in clinical trials, in a severe mouse model of DMD, the D2.mdx mouse, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression [~]10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease is observed. Surprisingly, a lethal acceleration of cardiac disease progression occurs with one of the micro-dystrophins, while the second appears to benefit the heart. The detrimental impact on the heart in the first case appears to be caused by the high levels of micro-dystrophin in the heart resulting in competition between micro-dystrophin and utrophin at the cardiomyocyte membrane. While the significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown, it suggests that micro-dystrophin treatments may need to be carefully titrated to avoid high levels of expression in the heart.

pharmacology and toxicology↗

bioRxiv2022

Evaluation of the DBA/2J mouse as a potential background strain for genetic models of cardiomyopathy

The potential use of the D2.mdx mouse (the mdx mutation on the DBA/2J genetic background) as a preclinical model of the cardiac aspects of Duchenne muscular dystrophy (DMD) has been criticized based on speculation that the DBA/2J genetic background displays an inherent hypertrophic cardiomyopathy phenotype. Accordingly, the goal of the current study was to further examine the cardiac status of this mouse strain over a 12-month period. DBA/2J mice have been scrutinized for the presence of cardiac lesions, however, in the current study we find that DBA/2J mice contain equivalent amounts of left ventricular collagen as healthy canine and human samples. In a longitudinal echocardiography study, neither sedentary or exercised DBA/2J mice demonstrated left ventricular wall thickening or cardiac functional deficits. In summary, we find no evidence of hypertrophic cardiomyopathy, or any other cardiac pathology, and thus propose that it is an appropriate background strain for genetic modeling of cardiac diseases, including the cardiomyopathy associated with DMD.

physiology↗

bioRxiv2022

NOX4 inhibition promotes the remodeling of dystrophic muscle

The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced stage disease progression. The current study investigates targeting NAD(P)H oxidase (NOX) 4 as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 is elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduces fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreases the number of fibrosis-depositing cells (myofibroblasts) and restores the number of muscle-specific stem cells (satellite cells) to their physiological niche, thereby, rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 is sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies. SIGNIFICANCE STATEMENTMuscular dystrophies are progressive muscle diseases. Therapeutics capable of combating the severe fibrosis that replaces functional muscle in these devastating diseases is a major unmet clinical need, particularly for the treatment of older patients. The current work reveals that targeting NOX4 in dystrophic muscle promotes the beneficial remodeling of disease-burdened musculature. This is achieved by the removal of disease-causing cells, known as myofibroblasts, which results in reduced muscle fibrosis and rejuvenation of muscle regeneration. NOX4-targeting strategies, therefore, represent remodeling therapeutics capable of improving muscle disease caused by muscular dystrophy, and, likely, other muscle pathologies.

cell biology↗

bioRxiv2021

Filopodia powered by class X myosin promote fusion of mammalian myoblasts

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. Conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multi-nucleated mammalian muscle formation.

cell biology↗

bioRxiv2020

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1

In mammals, the circadian clock coordinates various cell physiological processes, including the inflammatory response. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the circadian clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-{kappa}B in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-{kappa}B subunit RELA in the U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude in these cells, its inhibition lengthened period length and caused amplitude phenotypes. NF-{kappa}B perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity. We show that RELA, like the clock repressor CRY1, potently repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA competes with coactivator CBP/p300 for binding to the transactivation domain of BMAL1. This mechanism is further supported by chromatin immunoprecipitation analysis showing that the binding sites of RELA, BMAL1 and CLOCK converge on the E-boxes of clock genes. Taken together, these data support a significant role for NF-{kappa}B in directly regulating circadian clock function and highlight mutual regulation between the circadian and inflammatory pathways.

molecular biology↗