MSS51 is a mitochondria-associated protein specific to skeletal muscle whose expression is decreased when myostatin is inhibited, suggesting a role in myostatin signaling.
Mss51−/− mice were resistant to diet-induced weight gain and showed increased glycolysis, β-oxidation, and oxidative phosphorylation, indicating MSS51 has a role in the regulation of skeletal muscle mitochondrial respiration and glucose and fatty acid metabolism
[5552]. When
Mss51 was knocked out in
mdx mice, they demonstrated larger myofibers, less fibrosis, and greater mitochondrial respiration. These mice showed improvement in running endurance but not in grip strength
[5552].
3. Emery–Dreifuss Muscular Dystrophy (EDMD)
Emery–Dreifuss muscular dystrophy (EDMD) is a neuromuscular disorder characterized by a combination of skeletal muscle weakness, joint contractures, and cardiac abnormalities
[5653]. EDMD is rare; it has a prevalence estimated at 3 to 4 cases per million. Falling under the spectrum of laminopathies, EDMD is caused by mutations in at least 10 genes encoding nuclear envelope proteins. The most common types are caused by mutations in emerin (
EMD) and lamin A (
LMNA)
[5653][5754][5855].
EMD has an X-linked recessive inheritance
[5855], it encodes emerin, a nuclear membrane protein found at the interface of the nucleus with the cytoplasm.
LMNA is inherited in an autosomal dominant fashion, and it codes for lamin A and C, which are type V intermediate filaments. The lamins polymerize under the inner nuclear membrane to form the nuclear lamina
[5956][6057].
EDMD patients have diverse clinical presentations, making it challenging to diagnose and manage
[5653][55][58][61]. Patients with EDMD have three main sets of symptoms, which consist of early contractures, progressive muscle weakness and atrophy, and cardiac abnormalities. Joint contractures are a hallmark symptom of EDMD, causing multi-joint stiffness, reduced mobility, and functional impairment, most often involving the neck, elbows, and heel. Spinal rigidity may also affect posture, flexibility, and swallowing
[5653][5855]. Progressive muscle weakness and wasting primarily affect the scapulo-humero-peroneal muscles in children, which impairs mobility and strength
[5653].
In the nucleus, emerin binds to lamin A, actin, and nuclear myosin, and as a complex, it regulates chromatin dynamics, acts as a mechanosensor for the nucleus, and participates in the regulation of gene expression
[6259][6360][6461][6562]. EDMD pathophysiology involves disruptions in the nuclear envelope, resulting in loss of nuclear membrane integrity, heterochromatin decondensation, nucleoplasm leakage, chromatin detachment from the nuclear lamina, pseudoinclusions, and nuclear fragmentation (
Figure 1)
[5653]. These defects contribute to cellular dysfunction, especially in tissues like cardiac and skeletal muscles that undergo mechanical stress
[5855]. Histologically, the heart demonstrates myocardial fibrosis and fibrolipomatosis, and skeletal muscle has dystrophic changes, including variation in fiber size, increased number of myofibers with centrally localized nuclei, increased endomysial connective tissue, and the presence of necrotic fibers
[5855].
EDMD Treatment
EDMD is a challenging and genetically complex disorder, necessitating ongoing research to advance our understanding of the disease and develop effective therapeutic strategies
[5855][6663]. Timely cardiac interventions, regular monitoring, and supportive care are also essential in improving the prognosis and quality of life for individuals with EDMD
[5653]. Currently, the therapeutic approaches for EDMD patients are limited to the alleviation of symptoms, slowing disease progression, and improving the overall quality of life
[5653]. Much like DMD and BMD patients, glucocorticoids are prescribed to limit inflammation
[5754]. Due to side effects associated with long-term use of glucocorticoids, anti-inflammatory and anti-fibrotic agents are being examined in mouse models for EDMD.
4. Facioscapulohumeral Muscular Dystrophy (FSHD)
Facioscapulohumeral muscular dystrophy (FSHD) is the third most prevalent type of MD, following myotonic dystrophy type 1 and DMD, estimated to affect 1 in 15,000 to 1 in 20,000 people
[6764]. FSHD is inherited in an autosomal dominant manner; however, approximately 30% are de novo cases. Additionally, there is a high frequency of somatic mosaicism
[6865]. FSHD typically presents with weakness in one or more of these facial muscles, the stabilizers of the scapula, or the dorsiflexors of the foot. Muscle weakness is slowly progressive and, with time, can involve the axial and respiratory muscles and those of the lower limbs
[6966].
Histopathological studies of human FSHD biopsies have shown that there are regenerating myofibers of varying sizes; inflammation and fibrosis that is both endomysial and perivascular were prominent, and there was significant fat replacement of the muscle fibers
[7067]. A two-year prospective study of FSHD patients found that inflammation was mild in the early stages, but its presence was related to an increased rate of muscle degradation and fat infiltration
[7168]. When examining RNAseq and microarray data from FSHD biopsies, it was noted that inflammatory genes have increased levels of expression
[7269]. FSHD myoblasts have increased sensitivity to oxidative stress and are prone to apoptosis because double homeobox protein 4 (DUX4) affects mitochondrial function.
DUX4 is the gene affected in FSHD patients
[7370][7471].
DUX4 is expressed in pre-implantation embryos, and Dux binding sites are found in the control regions of genes involved in early genome activation
[7269][7370]. DUX4 is important in early embryos for embryonic genome activation (EGA). DUX4 triggers this process and subsequently becomes inactive
[7572][7673][7774][7875][7976]. Postnatally, DUX4 is expressed at low levels in the testis and the thymus. Ectopic expression in skeletal muscle postnatally results in FSHD
[8077][8178][8279]. The human
DUX4 gene is located on chromosome 4 in a region known as D4Z4, which consists of 11 to more than 100 repeated 3300 DNA base pair segments. Each of the repeated segments in the D4Z4 region contains a copy of the
DUX4 gene; the copy closest to the end of chromosome 4 is called
DUX4, while the other copies are referred to as “DUX4-like” or
DUX4L [8380][8481]
5. Limb-Girdle Muscular Dystrophy
Limb-girdle muscular dystrophy (LGMD) is the largest group of muscular dystrophies. They were originally defined by the progressive wasting of skeletal muscles of the pelvic and pectoral girdles. These dystrophies show significant variation in the onset of the disease, degree of wasting and inclusion of cardiomyopathy, cardiac arrhythmias, and respiratory failure [8582]. There are 24 genetic subtypes based on disease phenotype and mutations: (i) sarcoglycan complex (LGMD2C-F); (ii) glycosylation/α-dystroglycan complex (LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2S, LGMD2T, LGMD2U, LGMD2Z); (iii) sarcomeric proteins (LGMD1A, LGMD1D, LGMD1E, LGMD2A, LGMD2G, LGMD2J, LGMD2Q, and LGMD2R); (iv) signal transduction (LGMD1C, LGMD2P, LGMD2W); and (v) membrane trafficking and repair (LGMD1C, LGMD1F, LGMD2B, LGMD2L). These disorders are relatively rare compared to DMD, with individual estimated prevalences of 0.01–0.60 cases per 100,000 persons [8683]. The rarity of these disorders limits the ability to carry out clinical trials to examine the effectiveness of therapeutic strategies discovered in mice [8784].
5.1. Sarcoglycanopathy
Sarcoglycan consists of four subunits, α, β, γ, and δ, that form an integral membrane protein complex that is a subunit of the DGC in skeletal muscle. This protein complex is essential for the structural stability of the sarcolemma in both skeletal and cardiac muscle (
Figure 2). Genetic mutations of any sarcoglycan subunit are inherited in an autosomal dominant fashion and result in sarcoglycanopathy, which has a similar phenotype to DMD. In sarcoglycanopathy, or LGMD2, the average age at onset of muscle weakness is 6–8 years, with loss of ambulation at 12–16 years old. Similar to DMD, cardiomyopathy and respiratory complications following the loss of ambulation are commonly observed
[82][85][8886][89]. At the tissue level, muscle weakness is associated with chronic inflammation and the replacement of muscle fibers with fat depositions and fibrosis
[9087].
Figure 2.
Schematic of the etiology of subgroups of LGMDs.
5.2. Dystroglycanopathies
Dystroglycanopathies are autosomal recessive dystrophic disorders with variability in the age of onset and degree of muscle wasting. α- and β-dystroglycan form an integral membrane protein complex that links the basement membrane to the cytoplasmic dystrophin protein. α-dystroglycan binds to ECM and synaptic proteins and is dependent on heavy glycosylation . Dystroglycanopathies are primarily linked mutations in glycosyltransferases that target α-dystroglycan, including fukutin-related protein (/LGMD2I) , fukutin (/LGMD2M), -like-acetylglucosaminyl transferase (), protein-O-mannosyltransferase 1 (/LGMD2K), protein-O-mannosyl transferase 2 (/LGMD2N), protein-O-mannose-1,2-N-acetylglucosaminyltransferase 1 (/LGMD2O), dolichyl-phosphate mannosyltransferase polypeptide 3 (), and isoprenoid synthase domain containing (/LGMD2U) [90][91][92][93][94][95]. A rare subset of dystroglycanopathies includes mutations in the dystroglycan gene (DAG1). These mutations cluster in the N-terminal domain of α-dystroglycan or in the domains that stabilize the α/β-dystroglycan interface . Though these milder forms of muscular dystrophy are classified as LGMD based on their progressive muscle wasting in the extremities, more severe congenital muscular dystrophies are associated with mutations in glycosyltransferases targeting α-dystroglycan that result in a non-progressive muscle weakness that is observed at birth. Most notable among these are Walker–Warburg (WWS) and muscle eye brain syndromes (MEB), which present with an earlier onset of muscle weakness and include complex eye and brain disorders [97][98]. A rare subset of dystroglycanopathies includes mutations in the dystroglycan gene (DAG1). These mutations cluster in the N-terminal domain of α-dystroglycan or in the domains that stabilize the α/β-dystroglycan interface [99]. Though these milder forms of muscular dystrophy are classified as LGMD based on their progressive muscle wasting in the extremities, more severe congenital muscular dystrophies are associated with mutations in glycosyltransferases targeting α-dystroglycan that result in a non-progressive muscle weakness that is observed at birth. Most notable among these are Walker–Warburg (WWS) and muscle eye brain syndromes (MEB), which present with an earlier onset of muscle weakness and include complex eye and brain disorders [100][101].
The most common form of dystroglycanopathy, LGMD2I, is caused by mutations in
.
The most common form of dystroglycanopathy, LGMD2I, is caused by mutations in ; it occurs at a frequency of 1 in 250,000 in the U.S. The use of corticosteroids to reduce fibrosis and muscle loss has shown promise with LGMD2I. Mice that recapitulate the LGMD2I phenotype through mutation of have reduced pathology and muscle degeneration when treated with prednisolone, though it did not improve muscle strength. However, in combination with the bisphosphonate, alendronate, which is normally administered to limit osteoporosis, the level of muscle damage was lessened, and muscle function was improved . Under both conditions, the level of glycosylated α-dystroglycan increased, suggesting a mechanism other than immunosuppression for injury reduction.
6. Congenital Muscular Dystrophy
6.1. LAMA2-Related Muscular Dystrophy
MDC1A patients display hypotonia and muscle wasting at birth and with progressive spinal deformities and contractures of the major limb joints in early childhood
[10299][103100]. These patients can suffer from neuropathies under the age of 1 and can experience cerebral atrophy and seizures in older individuals
[104101][105102][106103]. In severe cases, the loss of muscle strength complicates swallowing and breathing, and death due to respiratory insufficiency can occur in the first decade, though their usual life expectancy is the thirties
[107104][108105].
Inflammation and fibrosis play prominent roles in the early onset of muscle weakness in MDC1A
[109106]. Individuals experience a burst of inflammation in their skeletal muscle as early as a couple of months after birth. This leads to the expansion of the interstitial ECM between myofibers, resulting in fibrosis. Unlike DMD, where fibrosis builds over time with repeated rounds of muscle breakdown and repair, fibrosis in MDC1A appears early and is maintained
[109106].
Fibrosis in MDC1A muscle has been linked to the TGF-β and renin-angiotensin signaling pathways. TGF-β is a primary regulator of fibrotic deposition via its roles in promoting the expression of ECM proteins and the conversion of satellite cells to a fibroblastic lineage
[16][110107].
Therapeutic strategies designed to reduce fibrosis in MDC1A patients have largely focused on reducing TGF-β levels. One approach that has shown promise is the AT
1R blocker losartan. Though initially approved by the FDA to control hypertension, it effectively reduced fibrosis in the
dyW/dyW and
dy2J/dy2J by disruption of Ang II signaling through AT
1R, which inhibits the conversion of TGF-β to its active form
[111108][112109][113110][114111]. Losartan treatment also decreased ERK phosphorylation and fibrosis in
dyW/dyW mice
[111108].
The FDA-approved serine/threonine kinase inhibitor vemurafenib effectively ameliorates fibrosis in
dy3K/dy3K mice
[115112]. Treatment resulted in a reduction in TGF-β and mTORC1/p70S6K signaling. Much like losartan, vemurafenib was able to block the pro-fibrotic pathway but was unable to promote muscle repair. This raises concerns about its use as a solo therapy.
In addition to blocking the TGF-β signaling pathway that directly induces fibrosis and inflammation, other research groups have explored the therapeutic benefits of blocking secondary events associated with muscle pathology in MDC1A patients and mouse models. Perhaps the most promise has been reported with omigapil, an inhibitor of the pro-apoptotic protein, Bax, and the GAPDH-Siah apoptosis cascade
[116113][117114]
The pathology in
Lama2-deficient mice appears to be exacerbated by the upregulation of lysosome-associated degradation pathways in the muscle
[118115][119116]. Consistent with this, inhibition of the ubiquitin-proteasome system by treatment of
dy3K/dy3K mice with MG-132 or bortezomib increased muscle strength and mobility and reduced muscle fibrosis while extending the mouse’s lifespan
[118115][120117]. In wildtype mice, Lama2 acts as an inhibitor of autophagy through the inhibition of the class III PI3K Vps34.
6.2. Collagen VI-Related Dystrophies
ColVI is a beaded fibril consisting of three chains encoded by
COL6A1,
COL6A2, and
COL6A3. The ColVI collagen fibers form a mesh-like network associated with the ECM of basement membranes, where it can modify the nature of the structure based on binding to glycoproteins, proteoglycans, and glycosaminoglycans
[121118]. They serve important roles in protecting cells from compressive forces while facilitating the interaction between cells and ECM proteins. Further, ColVI participates in the regulation of autophagy through direct interaction with integral membrane proteins on adjacent cells (β1 integrin, TEM8, and VEGFR2)
[122119][123120][124121].
Mutations in
COL6A1,
COL6A2, and
COL6A3 lead to a spectrum of congenital muscular dystrophies that range from UCMD at the severe end of the spectrum to Bethlem myopathy at the mild end
[121118]. UCMD patients experience congenital hypotonia and contractures in joints of the extremities.
The histopathology associated with muscle weakness in ColVI-related dystrophies includes muscle fibers of various sizes and a significant increase in interstitial fibrosis
[125122]. As with MDC1A, fibrosis accumulation is immediate and does not build with time, as seen with DMD. Using
Col61A-deficient mice as a model, fibrosis was found to be related to overactivation of mesenchymal stem cells that have been converted to the fibroblast lineage
[125122].
7. Conclusions
Advances in genetic, genomic, and proteomic technologies have resulted in an acceleration in the unraveling of the molecular mechanisms underlying the histopathology of more than 50 types of muscular dystrophy and opened the door to an exploration of alternative therapeutic approaches to glucocorticoid suppression of fibrosis, fat accumulation, and chronic inflammation associated with most muscular dystrophies. Strategies with broad promise are those that reduce proinflammatory NF-κB and TNF-α signaling. Studies in mouse models have shown success in ameliorating the pathology upstream of their expression through antioxidants (e.g., COX enzyme inhibitor)
[126123], suppression of the inflammasome (e.g., P2X7 antagonists)
[127124] or directly through the inhibition of NF-κB activity (e.g., vamorolone)
[128125]. Alternatively, inhibition of the Akt/mTOR signaling pathway by rapamycin suppresses the immune response to injury by reducing the proliferation of immune cells
[129126]. This has shown potential for ameliorating fibrosis in
Lmna- and
Ftkn-deficient mice
[10198][130127][131128][132129]. Recent reports predict that direct suppression of fibrotic deposition in fibroblasts at the site of injury by disrupting uPA/uPAR signaling via Serp-1 represents a strategy downstream of chronic inflammation to reduce fibrosis
[133130][134131][135132].