Treatment and Management of Muscular Dystrophies

Dystrophin Protein

The normal DMD gene creates a 14-kb dystrophin mRNA that encodes dystrophin, a 427-kDa protein. Dystrophin localizes to the subsarcolemmal region in skeletal and cardiac muscle and composes 0.002% of total muscle protein ( ; ; ). Dystrophin binds to the cytoskeletal actin and to the cytoplasmic tail of the transmembrane dystrophin-glycoprotein complex (DGC) protein β-dystroglycan, and through this to α-sarcoglycan, thus forming a link from the cytoskeleton to the ECM (see Fig. 20.1 ). The dystrophin protein is also found in brain, smooth muscle, and retina.

Primary and Secondary (Downstream) Events

Muscle cell death (by apoptosis and necrosis) in the muscular dystrophies is conditional on endogenous biochemical mechanisms and reflects a propensity that varies between muscles and changes with age ( Fig. 20.2 ) ( ). Although dystrophin deficiency is the primary cause of DMD, multiple secondary pathways are responsible for the progression of muscle necrosis, the abnormal fibrosis, and the failure of regeneration that results in a progressively worsening clinical status. The literature is rich in evidence supporting oxidative radical damage to myofibers ( ; ; ; ), inflammation ( ; ; ; ; ; ; ; ), abnormal calcium homeostasis ( ; ; ; ; ; ), myonuclear apoptosis ( ; , ; ; ; ; ), abnormal fibrosis, and failure of regeneration ( ; ; ; ; ; ; ; ; ). The following is a brief summary of the current understanding of how these processes occur.

Mechanical Membrane Fragility

Dystrophin is a link between the intracellular cytoskeleton and the ECM. The carboxy-terminal of dystrophin is attached to the sarcolemma, the surface membrane of striated muscle cells ( ; ; ; ), binding to β-dystroglycan ( ) and through this to other dystrophin-associated glycoproteins and to α-dystroglycan, which links the sarcolemma to the ECM ( ). When dystrophin is not present, the disconnection of contractile proteins from β-dystroglycan results in loss of β- and α-dystroglycan and the DGC from the sarcolemma. This disruption results in membrane fragility and abnormal permeability, particularly to calcium ions.

Abnormal Permeability to Calcium and Chronic Increase in Intracellular Calcium

Cumulative evidence points to elevated intracellular Ca ²⁺ homeostasis being a cause or facilitator to the development of muscle weakness in muscular dystrophies (Andersson et al., 2012; Goonasekera et al., 2011; Pal et al., 2014). Abnormal Ca ²⁺ handling may be related to direct dystrophin regulation of mechanosensitive transient receptor potential (TRP) channels ( ; ), as well as abnormal intracellular Ca ²⁺ cycling ( ; ; ).

The deregulation of Ca ²⁺ channels is seen in abnormal function of voltage-insensitive or “stretch-activated” Ca ²⁺ channels, a subfamily of the TRP channels ( ). These stretch-activated channels are abnormally active under mechanical stimulation in myotubes of mdx mice (murine model of DMD), resulting in an increase in intracellular calcium ( ; ; ).

The L-type, voltage-gated Ca ²⁺ channels also appear to be abnormal in the absence of dystrophin; it has been shown that the Ca ²⁺ currents in response to an action potential are much smaller in mdx mice than in normal controls. A disrupted direct or indirect linkage of dystrophin with these channels may be crucial for proper excitation-contraction coupling, initiating Ca ²⁺ release from the sarcoplasmic reticulum.

The abnormal intracellular Ca ²⁺ levels result in abnormal activation of Ca ²⁺ -activated proteases (i.e., calpain) with subsequent abnormal degradation of intracellular proteins, which probably contributes to the abnormal functioning of the calcium leak channels ( ). Cumulative preclinical evidence shows that chronic eccentric exercise worsens the abnormalities in calcium homeostasis in mdx mice ( ), which supports the clinical observation that eccentric exercises in DMD are deleterious and exacerbate muscle weakness ( ; ). Calcium also accumulates in mitochondria, contributing to cell dysfunction by affecting energy production (Dubinin et al., 2020; ).

Abnormal Immunologic Response

Dystrophic skeletal muscle is characterized by a persistent inflammatory response, with muscle degeneration, regeneration, and fibrosis. The lack of dystrophin in myofibers leads to contraction-induced membrane damage with release of cytoplasmic contents and self-sustaining stimulation of innate immunity. Proinflammatory cytokines induce recruitment of T and B cells and generation of an adaptive immune response in the muscle milieu. This proinflammatory microenvironment is often superimposed on the neutrophil and macrophage infiltrations induced by successive courses of myofiber degeneration and regeneration. In contrast to the full resolution of a single bout of inflammation and repair of normal muscle, dystrophin-deficient muscle loses the bout effect. Neighboring fibers or groups of fibers enter the necrotic stage at different times in the 2-week degeneration/regeneration cycle (asynchronous regeneration), thus sustaining a chronic inflammatory state, which, in turn, creates a more proinflammatory environment with activation of innate immune pathways, and evidence of increased antigen presentation (Dadgar et al., 2014; ).

Many of these immune response pathways are known to be blocked by the glucocorticoids prednisone and deflazacort, drugs that have been shown to slow progression of the disease, improve muscle strength and motor function, and prolong survival. Other therapeutic immunomodulatory approaches are being developed to alter the natural course of the disease and could be an adjunct treatment for other therapies such as gene and exon skipping therapies.

Abnormal Signaling Functions

Mounting evidence shows that the dystroglycan complex has important muscle cell signaling functions, and its integrity is essential for muscle cell viability ( ). These functions include transmembrane signaling (through β-dystroglycan), docking of signal transduction molecules (i.e., caveolin-3), and interaction with or regulation of other transmembrane complexes (i.e., integrins). When the dystroglycan complex is disassociated from the sarcolemma, there is a disruption of the cell signaling involved in regulating apoptosis ( ) and in the metabolism of reactive oxygen species ( ; ; ; ; ; ).

Another abnormality of cell signaling is likely the underlying explanation for the “vascular” theory of DMD pathogenesis, supported in the past by morphologic evidence of muscle fiber group necrosis occurring very early in the disease, presumably secondary to ischemia. Dystrophin absence results in mislocalization and reduction of neuronal nitric oxide synthase (nNOS) in dystrophic muscle, affecting smooth vessel vasodilation in response to alpha-adrenergic stimuli in exercise ( ), resulting in muscle ischemia ( ). Dystrophin-associated α-syntrophin appears to be essential for the membrane localization of nNOS ( ).

Abnormal Fibrosis and Muscle Regeneration

The chronic inflammation in DMD leads to a shift in balance from myogenic pathways resulting in successful muscle regeneration toward fibrogenic pathways, culminating in the loss of muscle fibers and their replacement with fatty-fibrotic matrix. The gradual accumulation of fibrosis in skeletal muscle dysregulates contractile function and is directly related to loss of motor function in DMD. Fibrosis also becomes a physical barrier that prevents normal blood flow to the muscle and the delivery of gene therapy or antisense-oligonucleotides to the muscle fiber. Thus, understanding the fibrotic process in DMD has become important to identify new therapeutic targets that could complement anti-inflammatory as well as dystrophin delivery therapies ( ).

Fibrosis (the excessive deposition of endomysial and perimysial ECM) is a known secondary phenomenon to chronic muscle inflammation and fiber degeneration in DMD ( ). Many signaling pathways modulate fibrotic progression in dystrophic skeletal muscle, and these provide important therapeutic targets to reduce fibrosis in DMD and other muscular dystrophies. The primary profibrotic signal is transforming growth factor-beta (TGFβ) (Ceco et al., 2013). High expression of TGFβ is evident in dystrophic muscle ( ) and in serum samples of individuals with DMD ( ). TGFβ is stored in the ECM as a complex with latent TGFβ-binding proteins (LTBPs). Activation of TGFβ requires its release from the complex by proteases into the ECM. Polymorphisms in the LTBPs have been associated with worse prognosis and increased fibrosis in DMD (Flanigan et al., 2013). Thus, blocking the cleavage of LTBP4 is a potential therapeutic target for preventing the excessive release of active TGFβ ( ). Once TGFβ is released, it can act through its canonical pathway of binding to the TGFβ receptor to mediate SMAD signaling, which upon nuclear entry leads to activation of a profibrotic transcriptional program that includes the production of collagen I (Ceco et al., 2013). Noncanonical pathways are also activated, including mitogen-activated protein kinase (MAPK) pathways and c-abl that, in turn, enhance transcription of profibrotic signals, including TGFβ itself, as part of the positive feedback in fibrosis ( ). Other identified therapeutic targets include TGFβ downstream profibrotic pathways, like the renin-angiotensin system (Sun et al., 2009) and growth factors, like CTGC, found to be upregulated in dystrophic animal models and patients (Sun et al., 2008).

Interestingly, the amount of fibrosis in DMD seems disproportionate to the clinical severity in the earlier stages of the disease. Observation of this fact spurred the idea that fibrosis may also occur independent from muscle necrosis, degeneration, and failed regeneration. Evidence suggests that both enhanced fibrinogenesis and decreased fibrinolysis ( ) are implicated in the development of muscle fibrosis in DMD. Serum from DMD patients also have increase basic fibroblast growth factor, a skeletal muscle cytoplasmic polypeptide that colocalized with dystrophin in the muscle membrane. Leaking of this growth regulator that stimulates connective tissue synthesis, induces satellite cell (SC) proliferation, and suppresses myogenic differentiation could contribute to early fibrogenesis ( ).

Dystrophin and Its Role in Satellite Cells

The cycles of segmental degeneration and regeneration of the dystrophin-deficient muscle fibers trigger SC recruitment for regeneration. During regeneration of dystrophic muscles, decisions regarding SC fate are regulated by intrinsic mechanisms and extrinsic signals (Almada et al., 2016). Dystrophin deficiency in SCs affects cell polarity and self-renewal. This results in asymmetric cell division, which in turn alters normal muscle repair mechanisms (Chang, Chevalier, & Rudniki, 2016). Exhaustion of SC pool has been a concern in DMD, as it was thought that the chronicity of this degeneration-regeneration process would result in SC pool depletion over time and result in failed regeneration (Heslop et al., 2000). However, it is known that dystrophic SCs are initially increased in muscle biopsies of young DMD patients when compared with matched normal controls (Bankole et al., 2013). Recent preclinical studies in different murine models of muscular dystrophy have shown that dystrophic mice retain their SC pool. Furthermore, the number of activated SCs is elevated and the dystrophic muscle retains its ability to form new muscle fibers, compatible with active regeneration and pool maintenance. However, this regenerative process is incomplete, with a possible defect in the maturation characteristics that could be determinant to the dysfunction of these fibers (Ribeiro et al., 2019). These new findings support efforts to improve late muscle regeneration as therapeutic approaches, even in advance disease.

Duchenne Muscular Dystrophy

Neuromuscular Involvement

Muscle fiber necrosis with elevated muscle calcium levels and a high serum creatine kinase (CK) enzyme level can be found in infancy in patients with DMD ( ), but the clinical manifestations are typically not recognized until at least age 3 years. This discrepancy represents a potential therapeutic window in which early interventions could theoretically prevent or delay the onset of symptoms. Walking often begins later than in normal children, and affected boys experience more falling than expected. Gait abnormality often becomes apparent by age 3 to 4 years, leading to clinical evaluation. Muscle weakness presents initially in neck flexor muscles, with power being less than antigravity. As a result, the child turns on his or her side when getting up from a supine position on the floor, which is the initial sign of the Gowers maneuver. Hypertrophy of calf muscles becomes prominent by age 3 or 4 years ( Fig. 20.3 ). Hypertrophy of other muscles, including the vastus lateralis, infraspinatus, deltoid, and less frequently the gluteus maximus, triceps, and masseter muscles, may also develop. Muscle mass is usually decreased in later stages in the pectoral, peroneal, and anterior tibial muscles.

Because of hip girdle weakness, untreated patients exhibit the Gowers sign by age 5 or 6 years. The patient assumes a locked-leg, buttocks-first position followed by pushing off the floor with the hands, literally pushing the trunk erect by bracing the arms against the anterior thighs. Patients also tend to rock from side to side when walking, producing the waddling gait that is typical in older boys with the disease. Climbing stairs also becomes difficult with disease progression, and eventually distal muscles of the arms and legs become weak.

Accelerated deterioration in strength and balance often results from intercurrent disease or surgically induced immobilization. A wheelchair is required when ambulation is no longer possible, typically near the end of the first decade in untreated DMD and about 3 to 10 years later in steroid-treated DMD. After loss of ambulation, contractures become more pronounced in the lower extremities; they also soon involve the shoulders, and kyphoscoliosis may develop. Cardiac and respiratory involvement often occurs in this later disease stage as well.

Adolescent patients manifest increasing weakness and are unable to perform routine daily tasks with their arms, hands, and fingers. The head may progressively flex forward as extensor neck muscles lose strength. Lower facial muscles may be involved in the advanced phase.

Clinical Characteristics of Becker Muscular Dystrophy

BMD is present in 3 to 6 per 100,000 male births ( ). The onset of weakness is later than in the Duchenne type, usually seen after age 7 years and often in the second decade; the disease is also marked by lordosis, calf hypertrophy, and other features of DMD of a milder phenotype ( Fig. 20.4 ). The course is prolonged into adulthood, often with a normal life span. Ambulation is typically maintained beyond age 30 years. CK level is usually between 2000 and 20,000 U/L but may be in the normal range for mildly affected males. Because these patients can be asymptomatic for decades, they commonly are misdiagnosed as having liver disorder when routine laboratory values show elevated transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), which are elevated in parallel with serum CK levels. At times, the BMD diagnosis is made after many years of gastrointestinal follow-up for elevated transaminases, until someone thinks of measuring the CK, which comes back extremely elevated. Pseudohypertrophy, proximal hip weakness resulting in the Gowers sign, and electrocardiogram (ECG) abnormalities associated with DMD are also common to BMD.

Chromosome Xp21 Microdeletion Syndromes

The Xp21 microdeletion syndromes are a series of syndromes that include DMD, Aland Island eye disease, adrenal hypoplasia, glycerol kinase deficiency, retinitis pigmentosa ( RP3 ), mental retardation ( MRX1 ), and ornithine transcarbamylase deficiency. The combination of these conditions led to the discovery of the DMD gene on Xp21 ( ).

Female Duchenne Muscular Dystrophy D Carriers and Manifesting Carriers

Carrier females are heterozygous with a normal DMD gene on one X chromosome and a mutant gene on the other. More than 90% of female carriers are asymptomatic. However, variable degrees of muscle weakness may be seen with skewed X-inactivation, in which more than half of the mutant X chromosomes are operant in muscle cells. Such cells are prone to degeneration. If a large number of abnormal muscle fibers are present in a given muscle, that muscle may display weakness. The degree of strength from one muscle to another may vary from normal strength to significant weakness.

Signs and symptoms of female dystrophinopathy include muscle weakness, myopathic findings on muscle biopsies, elevated CK levels, and partial absence of dystrophin in muscle ( ). Symptoms manifest in about one fifth of DMD carriers ( ). The diagnosis should be considered in women with elevated CK levels and muscle weakness, even in the absence of a positive family history for DMD ( ).

DMD carriers appear to have an increased incidence of cardiomyopathy. Studies have shown an incidence of asymptomatic cardiomyopathy ranging from 8% to 48% in adult DMD carriers ( ; ; ) and from 0% to 15% in DMD carrier girls under age 16 years ( ).