Duchenne Muscular Dystrophy Model: Advances in Rat Research

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration and weakness. While mouse models have traditionally been used to study the disease, rats offer physiological advantages that make them increasingly valuable for research. Their larger size allows for more precise physiological assessments, making them an important tool in preclinical studies.

Advancements in genetic engineering have enabled the development of rat models that closely mimic human DMD pathology, providing new insights into disease progression and potential therapies.

Genetic Alterations in Laboratory Rats

Genetically modified rat models have significantly advanced the study of Duchenne muscular dystrophy (DMD), offering a more physiologically relevant system for investigating disease mechanisms and treatments. Unlike mouse models, which exhibit a milder phenotype due to compensatory mechanisms, rats more closely replicate the severe muscle degeneration seen in human patients. This has been achieved through gene-editing technologies such as CRISPR-Cas9 and TALENs, which allow targeted disruption of the dystrophin gene, the primary defect in DMD.

A widely used DMD rat model involves deleting exon 23 in the Dmd gene, resulting in a complete loss of functional dystrophin. This mutation mirrors genetic defects in human patients, leading to progressive muscle wasting and fibrosis. These rats exhibit early-onset muscle weakness, increased serum creatine kinase levels, and histopathological changes resembling human pathology. Their larger body size facilitates detailed physiological assessments, including in vivo muscle force measurements and longitudinal disease progression studies, which are challenging in smaller rodents.

Beyond exon 23 deletions, researchers have introduced additional genetic modifications to refine DMD rat models. Some models disrupt dystrophin-associated proteins, such as utrophin, to better understand compensatory pathways, while others incorporate humanized mutations to evaluate gene therapy approaches. These refinements have been instrumental in testing exon-skipping therapies, CRISPR-based gene correction, and pharmacological interventions aimed at stabilizing muscle integrity. The ability to generate precise genetic alterations in rats has also enabled the study of modifier genes that influence disease severity, shedding light on potential therapeutic targets beyond dystrophin restoration.

Skeletal Muscle Pathology

The muscle degeneration observed in DMD rat models closely mirrors human pathology, providing a valuable system for studying disease mechanisms and treatments. One of the earliest changes is the presence of centralized nuclei within muscle fibers, a hallmark of ongoing degeneration and regeneration cycles. This feature, commonly seen in human DMD biopsies, reflects an attempt to compensate for dystrophin deficiency, though these efforts ultimately fail to prevent long-term deterioration. Unlike mice, which often exhibit milder pathology, DMD rats develop extensive fibrosis and fatty infiltration, particularly in weight-bearing muscles such as the gastrocnemius and tibialis anterior.

Histological assessments reveal widespread myofiber necrosis, characterized by fragmented sarcolemmal membranes and infiltration of non-contractile tissue. Structural instability results from the absence of dystrophin, a cytoskeletal protein responsible for linking the actin cytoskeleton to the extracellular matrix. Without this support, muscle fibers experience increased mechanical stress during contraction, leading to repeated cycles of damage and repair. Over time, the regenerative capacity of satellite cells becomes exhausted, contributing to muscle function decline. Staining techniques such as Masson’s trichrome and Picrosirius Red confirm significant collagen deposition, reinforcing the translational relevance of these models.

Functional assessments further illustrate disease pathology. Force contraction studies using ex vivo muscle preparations show marked deficits in twitch and tetanic force production, particularly in fast-twitch muscles prone to dystrophic changes. Electrophysiological recordings indicate altered excitation-contraction coupling due to disruptions in calcium homeostasis, a well-documented consequence of dystrophin deficiency. Calcium dysregulation activates proteolytic enzymes such as calpains, contributing to myofibrillar degradation. Longitudinal studies show these deficits worsen with age, paralleling the progressive decline in ambulation and strength observed in human patients. The ability to track these changes over time has been instrumental in evaluating therapies aimed at preserving muscle function.

Cardiac and Respiratory Features

DMD rat models exhibit pronounced cardiac and respiratory impairments that closely resemble human complications. One of the most significant cardiac manifestations is dilated cardiomyopathy, characterized by ventricular dilation and reduced contractility. Echocardiographic assessments reveal a gradual decline in ejection fraction and fractional shortening, mirroring the systolic dysfunction observed in DMD patients. This deterioration stems from dystrophin loss in cardiomyocytes, leading to structural instability and increased susceptibility to mechanical stress. Over time, myocardial fibrosis develops, particularly in the left ventricle, as evidenced by Picrosirius Red staining. Fibrotic remodeling contributes to conduction abnormalities, increasing the risk of arrhythmias, a common cause of mortality in DMD.

Respiratory function is also affected, with progressive diaphragm weakness leading to impaired ventilation and reduced lung capacity. Pulmonary function tests demonstrate declining tidal volume and minute ventilation, consistent with the restrictive lung disease seen in human patients. Dystrophin loss in diaphragm muscle fibers increases susceptibility to contraction-induced injury, leading to fibrosis and diminished force output. Over time, respiratory muscle weakening contributes to hypoventilation, particularly during sleep, increasing the risk of nocturnal hypoxia and respiratory infections. This decline in respiratory function is a major factor in DMD-related morbidity, often necessitating mechanical ventilation in later disease stages.

Neuromuscular Assessments

Neuromuscular function assessments in DMD rat models provide a detailed understanding of disease progression and treatment efficacy. Behavioral tests, such as grip strength and treadmill endurance trials, reveal significant declines in motor performance as the disease advances. These deficits are particularly pronounced in weight-bearing muscles, reflecting progressive weakness. High-resolution motion capture systems have identified gait abnormalities, including shortened stride length and reduced limb coordination, aligning with clinical observations in human patients.

Electrophysiological techniques further highlight neuromuscular deterioration. In vivo electromyography (EMG) recordings show increased spontaneous activity, indicative of muscle fiber instability and ongoing degeneration. Nerve conduction studies reveal delayed motor response latencies, suggesting early peripheral nerve involvement. These disruptions in neuromuscular transmission are compounded by alterations in synaptic architecture at the neuromuscular junction (NMJ). Structural analyses using confocal microscopy have shown NMJ fragmentation and destabilization in dystrophic rats, contributing to impaired signal transmission and progressive motor control loss. The breakdown of NMJ integrity plays a significant role in functional decline, making it a critical target for therapies aimed at preserving neuromuscular connectivity.

Molecular Markers in Disease Analysis

Molecular markers in DMD rat models provide insights into disease progression and potential therapeutic targets. Biomarkers associated with muscle degeneration, fibrosis, and inflammation have been extensively studied to track disease severity and treatment efficacy. Elevated serum creatine kinase (CK) levels serve as a widely recognized indicator of muscle damage, reflecting increased membrane permeability due to dystrophin deficiency. In DMD rats, CK concentrations are significantly higher than in wild-type counterparts, particularly during early disease stages when muscle turnover is most active. While CK levels decline over time as muscle mass decreases, additional biomarkers such as lactate dehydrogenase (LDH) and myoglobin provide a more comprehensive assessment of disease status.

Beyond serum biomarkers, transcriptomic and proteomic analyses have identified key molecular pathways implicated in DMD pathology. Gene expression profiling of dystrophic muscle has revealed upregulation of fibrotic markers such as collagen types I and III, along with increased transforming growth factor-beta (TGF-β) expression, a key driver of extracellular matrix remodeling. Pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), further contribute to disease progression by exacerbating muscle degradation. Proteomic studies highlight alterations in calcium-handling proteins such as sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), which play a role in impaired excitation-contraction coupling. These molecular signatures provide valuable targets for pharmacological interventions aimed at reducing fibrosis, inflammation, and calcium dysregulation.