Molecular and Cellular Basis of Genetically Inherited Skeletal Muscle Disorders

Abstract

Defects in over 500 genes underlie a diverse collection of neuromuscular disorders (NMDs). By definition, NMDs are diseases that arise from defects in muscle or nerve. The subset of NMDs that impact skeletal muscle are referred to as “myopathies” and “muscular dystrophies”, and are due to mutations in genes encoding muscle proteins. Many of these genes encode proteins that provide structural stability or bolster membrane integrity, while others are involved in protein turnover, trafficking, and electrical excitability. In this review, the genetic basis and biological function of mutant proteins associated with myopathies will be discussed. In addition, pathomechanisms and treatment strategies for these disorders will be highlighted. Because the causal genetic defects are known in most cases, strategies that target the underlying molecular defect will likely be the most efficacious approach to therapies, and current strategies utilizing this approach will be briefly mentioned. Since the identification of the first gene associated with a neuromuscular disorder in 1987 to the current day, the field has made tremendous progress and has led in advancing gene therapeutics.

Introduction

Skeletal muscle cells are large and multinucleated, containing highly organized contractile proteins that interact with each other to generate force and allow movement of the body. Each multinucleated muscle cell is surrounded by a thin layer of specialized connective tissue called the basal lamina which serves as an intermediary between the cell and the reticular lamina. This connective tissue bolsters membrane integrity, transmits forces of muscle contraction, and communicates endocrine and paracrine signals to the cell. Each muscle cell is innervated from a single synapse of the motor neuron, with the contact point termed the neuromuscular junction, which is the site where muscle contraction is initiated. Neuromuscular junction signals are transduced to the contractile apparatus via a process called excitation contraction coupling (ECC), which is mediated through a specialized structure called the triad. Defects can arise at essentially all points of the contractile process, from the neuromuscular junction, to the triad, to the contractile apparatus itself, and to the specialized matrix-membrane contacts that maintain and preserve membrane integrity. When mutations arise, they can lead to devastating consequences for skeletal muscle, leading to impaired ambulation, compromised breathing, and in the most severe conditions early death. These muscle diseases are collectively referred to as myopathies and/or muscular dystrophies, depending on the underlying genetic cause and the morphological appearance of the abnormal muscle on biopsy. Disorders that primarily impact the neuromuscular junction are termed myasthenic syndromes. While the latter share many similarities with other primary muscle conditions; however, these disorders will not be discussed further in this review, as they have been recently presented in depth¹.

The discovery of the first gene linked to a muscle disease demonstrated that mutations in the DMD gene underlie Duchenne muscular dystrophy (DMD)^2,3, followed later by the discovery of dystrophin as its protein product⁴. This initial finding led to an explosion of information about novel muscle proteins linked to skeletal muscle disorders. Many of the discovered genes and their protein products were previously unknown and thus these discoveries lent new insights and understanding about normal muscle cell biology. Over three decades later, at least 500 genetic loci have been identified and linked to neuromuscular disorders, i.e. disorders which originate from defects in motor neurons (neuropathic origin) or skeletal muscles (myopathic origin). Most of these loci encode for proteins; however, a subset is due to repeat expansions that create toxic RNA (e.g. myotonic dystrophy type 1 and 2), while another set of diseases are caused by aberrant transcriptional activity of a key regulator of myogenesis (DUX4, in fascioscapulohumeral dystrophy types 1 and 2). This review will not cover the myotonic dystrophies⁵ or FSHD^6,7.

In this review, we summarize what is known about myopathies and muscular dystrophies caused by mutations in protein coding loci, with a focus on those that are most common, best understood and that provide the most insight into muscle biology. We will discuss the function(s) of these proteins in skeletal muscle and how mutations lead to disease.

Myopathies Linked to Dystrophin (Dystrophinopathies and Dystroglycanopathies)

Introduction to dystrophin and the dystrophin glycoprotein complex (DGC)

The dystrophin glycoprotein complex (DGC) is a multi-protein membrane complex comprised of intracellular, extracellular and transmembrane proteins^8,9. The complex can be divided into two main sub-complexes referred to as the sarcoglycans and the dystroglycans, which are linked together by sarcospan^10,11 and retained at the membrane by dystrophin⁹. There are six different sarcoglycan genes and one dystroglycan gene (DAG) that generates two different DAG proteins (αDAG and βDAG) after post-translational processing of a single polypeptide chain^12,13. The skeletal muscle sarcoglycan complex is comprised of four sarcoglycans (α,β,γ,δ).¹⁴ Dystrobrevin binds to the DGC, acts as a scaffold and provides additional stabilization; however, the DGC’s membrane association is not wholly dependent on dystobrevin.¹⁵

The DMD gene is the largest in the genome (2.2 Mb), which encodes a 14kb dystrophin mRNA. Six different promoters drive expression of dystrophin isoforms in skeletal, cardiac, smooth muscle and brain. These isoforms are named Dp426 (the primary skeletal and cardiac muscle isoform), Dp260, Dp116, Dp140, and Dp71. In skeletal muscle, dystrophin is enriched at costameres, which are sites where terminal Z-discs connect to the sarcolemma, and where force transmission occurs across the membrane¹⁶ (Figure 1).

Figure 1: Schematic of the costamere and proteins linked to the dystrophin glycoprotein complex (DGC).

a| The terminal Z disc of the skeletal muscle sarcomere attaches to the membrane at the costamere, where it links to a large protein complex called the DGC. b| On the intracellular side of the membrane, the DGC links to the actin cytoskeleton, microtubules and intermediate filaments via dystrophin protein (aqua). Dystropin is a 427kDa protein comprised of an N-terminal actin binding domain (ABD), 4 hinges, 24 spectrin repeats, a cysteine rich (CR) region that attaches to beta dystroglycan, and a C terminus. Dystrophin is a scaffold for several other molecules including neuronal nitric oxide synthase (nNOS), which attaches vis syntrophin (SYN), as well as ankyrin and dystrobrevin. c| The DGC is made of two membrane-associated subcomplexes, the dystroglycan (DAG) complex and the sarcoglycan complex (consisting of α, β, γ, and δ sarcoglycans), linked together by sarcospan (SSPN). d| Alpha dystroglycan (α-DAG) is the extracellular-facing DGC member in the basal lamina of the ECM. It is an ECM receptor that interacts with laminin, primarily through O-linked glycans on its mucin domain. e| α-DAG also associates with other matrix molecules such as nidogen and perlecan that along with collagens IV and VI, connect the basal lamina to collagens I and III in the reticular lamina.

Dystrophin has four main structural domains: 1) the N terminal region comprised of two actin binding domains^17,18; 2) a central rod domain (discussed more below); 3) a cysteine rich region that anchors dystrophin to beta dystroglycan¹⁹ and 4) a C terminus that scaffolds molecules like syntrophin²⁰ and dystrobrevin¹⁵. It also contains four proline-rich hinges that contribute to dystrophin’s flexibility. The central rod domain is comprised of 24 alpha helical spectrin-like repeats that confer dystrophin’s spring-like properties. The spectrin repeats serve other functions as well, including interaction (via repeats 1–3 and 10–12) with phospholipids in the sarcolemmal membrane²¹ and binding (via repeats 16–17) the adapter alpha syntrophin²², which help retain neuronal nitric oxide synthase (nNOSμ) at the sarcolemmal membrane^22,23. Neuronal NOS also interacts with syntrophin proteins bound to the C terminus of dystrophin. Spectrin repeats 4–15 and 20–23 have been shown to organize transverse microtubules²⁴.

The DGC serves three main roles in skeletal muscle fibers: 1) protecting the sarcolemmal membrane from stresses that arise during muscle contraction; 2) linking the intracellular cytoskeleton to the extracellular matrix and transmitting forces of muscle contraction to the tendon and 3) serving as a scaffold for signaling molecules such as syntrophin and Grb2.²⁵ Other proteins that associate with the DGC include filamin C²⁶, Ankyrin B²⁷, Synemin²⁸, aquaporin²⁹, syncoilin^30,31 and keratin 19³². Dystrophin is anchored to the membrane at its cysteine-rich C terminal region via beta dystroglycan, which in turn binds the C-terminus of alpha dystroglycan³³. Alpha dystroglycan is an important receptor for many basement membrane proteins such as laminin³⁴, agrin³⁵, nidogen^36,37 and perlecan³⁸. These associations help organize the ECM, contribute to the its structural stability and transmit forces of muscle contraction.

Defects in dystrophin and DGC members (dystrophinopathies and LGMDs)

Mutations in a number of DGC-linked genes cause muscular dystrophy or myopathy and are referred to as “dystrophinopathies” with a subset named “dystroglycanopathies”. The best characterized and most prevalent of these is Duchenne muscular dystrophy (DMD) due to mutations in DMD^3,39–41. DMD is on the X chromosome, so mutations primarily affect boys, with females primarily as asymptomatic carriers. The majority of pathogenic DMD mutations are deletions that occur within the central rod domain, and lead to frameshift with loss of RNA stability or a introduction of a premature termination codon.

Since the original discovery of the DMD gene^42,43, basic science investigations have provided new insights on the role of dystrophin and the DGC on muscle biology. A phenotypically milder disease called Becker muscular dystrophy (BMD) also arises from DMD mutations, but BMD mutations maintain the DMD reading frame and generate internally deleted, but functional dystrophin proteins. In-frame deletions that preserve the phasing of the repeats, as compared to ones that do not, result in dystrophins with enhanced sarcolemmal membrane protection, suggesting the concept that dystrophin protects the sarcolemma through its spectrin repeats⁴⁴. The majority of studies in the mdx mouse model of Duchenne have shown that loss or reduction of dystrophin leads to impaired membrane integrity and subsequent muscle cell degeneration^45–47, following by inflammation^48–51, regeneration⁵² and ultimately, muscle cell replacement by fat and fibrosis. Serum creatine kinase is elevated from birth, due to loss of dystrophin’s contribution to structural integrity of the sarcolemmal membrane.^45–47 The clinical progression of the dystrophinopathies has been previously described^53–55.

In addition to DMD and BMD, a class of muscular dystrophies referred to as limb girdle muscular dystrophies (LGMDs) result from mutations in genes associated with the DGC. LGMDs are progressive muscle wasting disorders that initially present with symptoms in the proximal musculature; however, weakness progresses to many muscles in the body, depending on the type of LGMD. They can be inherited in autosomal dominant or autosomal recessive fashion, and of the over 30 genetically defined LGMDs, a majority are linked to the DGC. Mutations in genes encoding four of the sarcoglycans (alpha⁵⁶, beta⁵⁷, delta⁵⁸ and gamma⁵⁹) cause autosomal recessive LGMD. Assembly of the sarcoglycan complex occurs in the golgi, starting with association between delta and beta sarcoglycans, followed by addition of alpha and gamma¹⁴. Null mutations in delta sarcoglycan prevent the sarcoglycan complex from forming, suggesting that it is a key organizer of the complex¹⁴. Null mutations in the other sarcoglycans tend to lead to a reduction, but not complete loss of the sarcoglycan complex.

Dystroglycan (DAG) is a key component of the skeletal muscle DGC which is also expressed in other tissues, where it assembles DGC-like complexes. In skeletal muscle, αDAG resides on the extracellular surface and serves as a receptor for many ECM proteins, most important of which is laminin (laminin 111/211) in the basement membrane. αDAG binds laminin in the central part of the molecule (mucin domain) which is glycosylated by several glycosyl transferases, each of which adds its unique sugar in order to build the O-linked moieties that are necessary for αDAG’s attachment to laminin. The first glycosyltransferases to add O-mannose to serine and threonine residues on αDAG are POMT1 and POMT2, which reside in the ER⁶⁰, followed by addition of GlcNAc by POMGNT2 and addition of GalNAc by β3GalNT2. Subsequently, this O-linked glycan is modified by ISPD (which attaches ribitol⁶¹). This core structure of three sugars (“M3 core”) is then phosphorylated on the O-linked sugar by a kinase named POMK (SGK196)^60,62 and further growth of the sugar structure takes place on this phosphate residue in the golgi. POMGNT1 and other glycosyl transferases such as FKRP, FKTN, TMEM5, and LARGE1 work together to generate the laminin binding motif⁶³, comprised of a repeating di-saccharide (-GlcA-β3-Xyl-α3)n⁶⁴ referred to as “matriglycan”⁶⁵. The specific roles of FKRP, FKTN and TMEM5 and the manner in which matriglycan is attached to the phosphate is still not known. The length of matriglycan is modulated by a sulfotransferase, HNK-1, which competes with LARGE1 by 3-O-sulfation of glucuronic acid⁶⁶. The presence of DAG’s N-terminal “DGN” domain is necessary for proper glycosylation of the mucin domain, which may be linked to N-linked glycosylation; however, further exploration is needed to elucidate the relationship between the presence of DGN and O glycosylation of the mucin domain. For additional review of dystroglycan glycosylation, please see⁶⁵.

Defects in dystroglycan and dystroglycan glycosylation (dystroglycanopathies)

Dystroglycanopathies share a common feature of impaired or absent αDAG glycosylation and encompass a spectrum of phenotypes, ranging from severe congenital muscular dystrophies with eye and brain involvement to milder “muscle only” presentations including subtypes of LGMD. Since αDAG is not restricted to skeletal muscle, and because it serves an important developmental role, loss or mutations in a subset of glycosyl transferases can result in severe CMDs with multi-organ involvement, affecting the eye and brain, such as Fukuyama CMD, muscle-eye-brain disease, and Walker–Warburg syndrome.^67–72 Of note, DAG mutations have been reported as an extremely rare cause of muscular dystrophy; it is assumed that most DAG mutations would not be compatable with life.

Defects in extracellular matrix (ECM) proteins

The sarcolemmal membrane attaches to the basal lamina (comprised of laminin α2, type IV collagen, heparin sulfate proteoglycan, agrin and perlecan), which is in turn is connected to collagen VI, which links the basement membrane to the reticular lamina (comprised mainly of collagens I and III). Mutations in genes encoding the three chains of the collagen VI fibril (COL6A1, COL6A2 or COL6A3) underlie autosomal dominant Bethlem or autosomal recessive Ullrich myopathy, characterized by muscle weakness and contractures. Mutations in COL6A2 can also lead to the milder LGMD R22 or LGMD D5. Most often, these mutations interfere with formation of the trimeric collagen myofibril, with severity dictated by which stage of myofibril assembly is interrupted^73–75. Laminins are also trimers, but only mutations in the α2 chain are linked to muscular dystrophy. Mutations in the LAMA2 gene lead to loss or reduction of laminin with corresponding clinical severity from mild (LGMD presentation) to severe (CMD presentation). While laminin 211 and collagen VI are extracellular matrix proteins, mutations share a common pathomechanism of increased myofiber apoptosis. Promoting myocyte survival is one potential treatment strategy for these muscular dystrophies; pre-clinical work has supported the efficacy of this approach, and one drug (omigapil) is currently in early-stage clinical trials.

Gene Therapeutics for DGC-linked diseases

Most myopathies and muscular dystrophies are amenable to gene replacement, antisense oligonucleotide-mediated exon skipping or gene editing approaches to therapy. Much of the focus in therapeutic development for the dystrophies has been on DMD, because this disease is the most prevalent. Because dystrophin can tolerate large, in frame mutations (as evidenced by the clinically milder BMD), most DMD-therapies have focused on altering the reading frame of the mRNA to change DMD mutations into BMD mutations. Modified antisense oligonucleotides (e.g. phosphorodiamidate morpholino oligomers or PMOs), can be systemically administered to skip single exons to reframe the mRNA, and three such therapies have been FDA approved (Exondys 51, Vyondys 53 and Viltepso) for mutations amenable for skipping exons 51 or 53. Additional PMOs are in development for other DMD exons. These exon skipping therapies must be administered by weekly infusion, do not target the heart, and result in only approximately 1% dystrophin re-expression. Adeno associated virus (AAV) has been used to deliver ASOs in murine studies with good success⁷⁶. New oligonucleotide chemistries are in development (e.g. PPMO), and these new formulations show improved uptake in skeletal muscle and heart^77,78. Exon skipping has also shown promise for gamma sarcoglycan deficiency⁷⁹ and in diseases in which mutations create pseudo-exons that can be skipped⁸⁰. Gene replacement therapies using AAV to deliver an engineered DMD transgene that retains approximately 30% of the native protein (referred to as mini- or micro-dystrophin) are in phase II and phase III trials, with promising preliminary reports. AAV-mediated gene replacement therapies are also in various stages of development for several of the autosomal recessive LGMDs and CMDs. For reviews of gene replacement therapies please see⁶³. CRISPR/Cas9 gene editing therapies are in preclinical development for DMD and clinical trials may initiate in the next few years^81–85. For review of CRISPR gene editing for neuromuscular disorders, please see Young et al.⁸⁶

Sarcomere pathologies

Introduction to sarcomeric proteins

The sarcomere is the fundamental unit of the myofiber and is the structure that ultimately generates force (Figure 2). It is composed of two primary components, the thin filament and the thick filament, embedded and tethered between two Z discs. The thin filament is composed primarily of skeletal muscle actin (encoded by ACTA1), with the addition of a group of proteins that regulate its length, dynamic interactions with the thick filament, and its stability. The thick filament is composed primarily of myosin, with different myosin isoforms found in distinct muscle fiber subtypes (for example, slow oxidative skeletal muscle vs fast glycolytic muscle). Muscle contraction is achieved when myosin slides along the actin thin filament, made possible by the regulated release of intracellular calcium (released from stores in the sarcoplasmic reticulum), which binds the troponin complex, changing tropomyosin’s confirmation to expose and enable myosin interaction with actin. In keeping with the importance of sarcomeric proteins in muscle contraction, mutations in components of both the thin and thick filaments lead to muscle disease. Also, and perhaps unsurprisingly because of the fact that the sarcomere ultimately performs the “end step” of muscle function, sarcomeric diseases have proven mostly refractory to therapy, and it is likely that gene correction or replacement-based strategies will be necessary to treat the majority of patients with these myopathies⁸⁷.

Figure 2 |. Schematic of a muscle cell and the proteins linked to the sarcomere Figure 2. Sarcomere structure in skeletal muscle.

a. Transmission electron micrograph of a longitudinal section of the sarcomere of zebrafish skeletal muscle. Z-disks are visible as electron-dense zig-zag vertical lines, M-band as a smooth dark line in the middle of the sarcomere, and the horizontal striations represent thick and thin filaments. The vacuolated areas are the triads, where excitation contraction coupling takes place. b. Schematic of the major protein components of the sarcomere. Thin filaments are composed of actin, nebulin, tropomyosin and the troponin complex with the following subunits: troponin T (TNNT, binds tropomyosin), troponin I (TNNI, binds actin), and troponin C (TNNC, binds the calcium ions). Thick filaments are composed of myosin and titin. Myosin consists of several domains: a head [two identical myosin heavy chains (MyHC), which bind actin], a neck [one pair of essential light chains (MyELC) and one pair of regulatory light chains (MyRLC)], and a tail. c. Schematic of a sarcomere in a relaxed state. d. Schematic of a sarcomere in contracted state. e. Schematic of a sarcomere during a defective contraction, which is a hallmark of thin-filament related nemaline myopathies. Mutations in nebulin (NEB) and actin (ACTA1) can lead to shorter thin filaments, defective actin assembly and dynamics, reduced force during muscle contraction, and lower sensitivity to calcium ions.

Defects in thin filament proteins that cause myopathies

Nemaline myopathy – the myopathy of the thin filament

Mutations in many components of the thin filament cause a subtype of congenital myopathy called nemaline myopathy⁸⁸. The most common cause of nemaline myopathy is recessive mutation in the NEB gene⁸⁹, with heterozygous mutation of the ACTA1 gene being second most common^90–92. While there are exceptions, the majority of patients with mutations in components of the thin filament present with a consistent clinical picture (non-progressive diffuse weakness, reduced muscle bulk, and selective involvement of the bulbar and axial musculature) and with shared features on muscle biopsy (myofiber hypotrophy, type I fiber predominance, myofibril disorganization, and the presence of aggregated Z disc material termed nemaline rods or nemaline bodies)⁹³.

Nebulin and NEB related NM

NEB encodes for nebulin, one of the largest proteins in the human genome, the function of which was a mystery for many years, resulting in the unique naming of the gene and its product⁹⁴. Nebulin is composed primarily of a repeat structure characterized by a series of actin binding domains interspersed with tropomyosin binding domains⁹⁵. The extreme N terminus extends to the end of the thin filament (where it interacts with tropomodulin), while the C terminus binds and embeds into the Z disc⁹⁴. A growing body of evidence supports that the main function of nebulin is to serve as a molecular ruler that dictates the length of the thin filament^96–99. Key data in this regard include the fact that loss of nebulin results in reduction of thin filament length, that across different species thin filament length correlates with the size of the nebulin protein, and that manipulating nebulin size (making it larger or smaller) correspondingly changes the length of the filament^96,100. Since muscle force is generated through the interaction between thick and thin filaments, there is a tight correlation between actin filament length and force generation; thus, loss of nebulin results in reduced contractile force and muscle weakness¹⁰¹. Nebulin has additional functions including regulating actin/myosin cross bridging (independent of its impact on thin filament length¹⁰²) and participating in signalling pathways critical for actin filament formation¹⁰³. The importance of these latter functions is less well established, and the consequence of their loss not known.

ACTA1 related NM

ACTA1 encodes skeletal muscle actin; polymers of ACTA1 form the core structure of the thin filament. Actin polymers interact with nebulin, tropomyosins, troponins, elements in the Z disk, and, critically, myosin (to enable muscle contraction)^104,105. Mutations in ACTA1 are the commonest dominant mutations found in congenital myopathies, and can produce a range of pathological changes in the muscle (with nemaline myopathy pathology the most common)⁸⁸. More than 200 different mutations in ACTA1 have been described¹⁰⁶. The majority of disease associated variants are missense changes that impair some aspect(s) of actin function, including assembly and stability of the actin filament, as well as potentially altering thin filament dynamics (calcium binding, sliding speed, etc). As with NEB loss of function, the majority of ACTA1 mutations are associated with reduced maximal force generation, which may result from reduced thin filament length in some instances¹⁰¹, and dysfunctional contractility in others¹⁰⁷. Of note, some mutations result in hypercontractility that causes a clinical phenotype associated with muscle stiffness¹⁰⁸.

There are currently no treatments for ACTA1-related myopathy, and few therapeutic targets. Case report level data has implicated L-tyrosine as a potential therapeutic for several nemaline myopathy subtypes¹⁰⁹, primarily for improving bulbar function, but studies in pre-clinical models have not supported its efficacy^110,111. Because myofiber smallness is a nearly invariant feature in muscle biopsies from ACTA1 patients, myostatin inhibition has been tested in a transgenic mouse model of the disease, with the result being increased muscle size and muscle force generation¹¹². Conversely, a similar examination of myostatin inhibition in nebulin deficient mice did not produce meaningful improvements¹¹³. Lastly, cardiac alpha actin is an intriguing target for ACTA1 disease. In addition to expression in the heart, ACTC is expressed in fetal skeletal muscle, and it functions similarly in terms of thin filament mechanics. Germline replacement of Acta1 with Actc in mice results in normal mice without evidence of muscle abnormalities¹¹⁴. Further, overexpression of Actc1 improves survival in one transgenic mouse model of dominant Acta1 mutation, though for unclear reasons does not promote improvement in a knock-in model harboring a different mutation¹¹⁵.

Other forms of Nemaline Myopathy

Mutations in TPM2, TPM3, TNNT1, TNNT3, TNNI2, and LMOD3 are all associated with nemaline myopathy and/or distal arthrogryposis (a syndrome of congenital joint contractures discussed below) and are regulatory components of the thin filament^116–121. TPM2 and TPM3 encode tropomyosin 2 (found in type 1 fibers) and tropomyosin 3 (found in type 2 fibers) respectively. Tropomyosins are coiled-coil proteins that bind along the length of the actin filament and both help stabilize the thin filament and participate in the calcium dependent switch between the relaxed and active state¹²². Mutations in TPM2 and TPM3, which typically impact tropomyosin dimerization and/or actin binding, generally result in either hypocontraction, associated with reduced calcium sensitivity and decreased actin-myosin sliding speeds, or hypercontraction, with higher calcium sensitivity and increased filament sliding¹²³. Of note, the hypercontraction mutations are more likely to be associated with joint contractures, and can present with a phenotype of arthrogryposis¹²⁴. TNNT1 encodes the slow skeletal muscle form of troponin; three troponin isoforms (T, C, and I) interact with tropomyosin and together form a complex that regulates the calcium sensitivity of muscle contraction. Interestingly, mutations in the I isoform (i.e. TNNT1) cause congenital myopathy¹¹⁸, while mutations in TNNT3 and TNNI2 are predominantly associated with arthrogryposis^125,126. LMOD3 encodes leiomodin 3, a member of the leiomodin protein group that are part of the tropomodulin family¹¹⁹. Tropomodulins in general are found at the barbed end of the actin filament where they interact with tropomyosins and “cap” the end of the thin filament, thus helping establish the length of the filament. LMOD3 in particular is a strong nucleator of actin polymerization, and may have a specific role in promoting and regulating thin filament formation.

The function of the remaining genes associated with nemaline myopathy (CFL2, KBTBD13, KLHL40, and KLHL41)^127–130 are less well understood. The latter three genes encode kelch domain containing proteins, and are substrate adaptors for the U3 ubiquitin ligase cullin-3. Evidence is emerging that they participate in the regulation of turnover of the core thin filament proteins. KLHL40, for example, interacts with both NEB and LMOD3, and may reduce their turnover by limiting their polyubiquitination¹³¹. KLHL41 binds and targets for ubiquination NRAP (nebulin anchoring protein). Overexpression of NRAP impairs myogenesis, while reducing NRAP levels in zebrafish lacking klhl41 rescues aspects of their myopathic presentation, suggesting a critical interplay between KLHL41 expression and activity, NRAP chaperone function, and muscle development and that KLHL41 mutation driven NM pathology¹³².

Myopathies of the thick filament

The core unit of the thick filament is the myosin hexamer. It is composed of two units of myosin heavy chains, two essential light chains, and two regulatory light chains. Myosin slides over the thin filament in an ATP dependent manner to generate a muscle contraction. There are 8 myosin heavy chains, 3 essential light chains, and 3 regulatory light chains that predominate in skeletal muscle¹³³. In addition, there are several regulatory proteins are associated with the thick filament, the most important of which from a muscle disease consideration is titin (TTN).

Disorders of the myosin heavy chain

Myosin heavy chains form homodimers that function as the main action component of the thick filament. They have a globular head domain that interacts directly with actin and has ATPase activity, and a distal rod domain important for dimerization and other protein-protein interactions. Of the 8 myosin heavy chains (MyHCs) found in skeletal muscle, mutations in 4 genes (MYH2, MYH3, MYH7, and MYH8) are associated with muscle disease^134–137. Mutations in MYH3 and MYH8 are associated with arthrogryposis, a condition characterized by multiple joint contractures present from birth. MYH3 encodes an embryonic form of myosin heavy chain, and dominant mutations cause a spectrum of severe joint contracture disorders that likely result from weakness and lack of limb movement during embryogenesis¹³⁵. MYH8 encodes a perinatal MyHC isoform, and dominant mutations cause a milder arthrogryposis syndrome called Trismus-pseudocamptydactyly syndrome (jaw contracture and unusual finger bending). MYH2 codes for myosin heavy chain IIA (found exclusively in Type IIA fibers) and mutations cause both dominant and recessive myopathies^134,138. Both forms have important involvement of the eye muscles; dominant mutations are associated with myopathy with rimmed vacuoles and a typically milder clinical presentation, while recessive mutations cause a congenital onset myopathy with absent type 2A fibers.

MYH7 mutations are the most common form of myosin related myopathy. MYH7 encodes a “slow” MyHC isoform expressed in type I skeletal myofibers and in cardiomyocytes. More than 200 dominant mutations have been identified that cause either skeletal and cardiac myopathy, with some rare patients manifesting both diseases¹³³. The skeletal myopathy is associated with a range of clinical phenotypes and biopsy findings, including the named syndromes of Laing distal myopathy and Myosin Storage Myopathy^137,139, and most commonly occurs with mutations in the distal rod domain. Clinical symptoms most typically start in the distal musculature, with the hanging big toe a common presenting sign, and are often mild. However, severe presentations, including those with congenital onset and failure to achieve ambulation, have been described¹⁴⁰.

Titin and TTNopathies

TTN encodes the giant myofilament protein titin, one of the largest and most complex proteins in the human genome. Titin spans from the Z-disc (at its N terminus) to the M line, so is essentially the length of one half of the sarcomere (approximately 1 μM). It is laid down early in development and is believed to be a template for sarcomere formation. Titin has myriad functions in skeletal muscle, the most important and well-studied of which are its roles as a molecular spring, as a generator of passive stiffness for the myofiber, and as a regulator of actin contractile force generation¹⁴¹. It also has kinase functions and has been implicated in signalling pathways. Unsurprisingly, given its large size and importance for muscle physiology, mutations in TTN are a frequent cause of skeletal myopathy, and may be the second most common cause of non-dystrophic muscle disease in childhood¹⁴². TTN is also one of the most frequently encountered causes of cardiomyopathy.

TTN mutations affecting skeletal muscle were first described in patients with a rare, mild dominant form of muscular dystrophy called tibial muscular dystrophy¹⁴³. An additional dominant form of TTNopathy, termed myofibrillar myopathy with early respiratory failure to reflect the clinical and histopathologic features, was subsequently described¹⁴⁴. More recently, and reflecting the increased ability to interrogate the TTN genomic locus affording by next generation sequencing, TTN has been identified as a cause of several recessive subtypes of congenital myopathy, including centronuclear myopathy and myopathy with cores. The clinical spectrum of patients with recessive TTNopathy is broad, ranging from early onset with delayed motor milestones and impaired ambulation, to later onset forms with mild/moderate diffuse weakness¹⁴⁵. At present, there are no therapies for the skeletal muscle manifestations of TTN mutations.

Introduction to the nuclear envelope

Just as structural proteins are essential to connect the extracellular matrix to the sarcolemma, myonuclei similarly have structural proteins that span the nuclear envelope and anchor the nuclear matrix (Figure 3). Specifically, the protein Lamin A/C (LMNA) connects the nuclear lamina on the inside of myonuclei with the inner nuclear membrane by interacting with emerin (EMD) and the SUN domain proteins, SUN1 and SUN2¹⁴⁶. These proteins then connect with the outer nuclear envelope and cytoskeleton via a family of proteins with a KASH domain termed nesprins (SYNE). This complex creates the linker of nucleoskeleton-and-cytoskeleton (LINC) that maintains myonuclear organization and structural integrity¹⁴⁷. The nuclear membrane serves to separate proteins in the sarcoplasm and nucleoplasm. While the nucleus requires this protective isolation, it also needs to communicate with the rest of the myofiber, exchanging proteins and RNA, for a variety of nuclear and cytoplasmic processes which act in concert. The nuclear pore complex (NPC), perhaps the largest protein complex in the cell (~125mDa), is responsible for the protected exchange of components between the nucleus and cytoplasm¹⁴⁸. The nucleocytoplasmic shuttling of proteins and RNA across the nuclear envelope via nuclear pore complexes (NPCs) further requires a family of nuclear transport receptors called importins to enter the nucleus, and exportins to exit. These receptors target specific cargo and facilitate transport through the NPC utilizing the hydrolysis of GTP within the nucleus via the small ras GTPase, Ran¹⁴⁸.

Figure 3: Schematic of a muscle cell and the proteins linked to the nuclear envelope.

A) Nuclear import and export through nuclear pore complexes requires transport receptors such as importins, exportins and transportins. These proteins shuttle cargo through the NPC along using a GTP gradient generated by RAN GTPase. B) The LINC complex connects lamins that compose the nuclear matrix on the inside of the nuclear envelope with via a family of proteins termed SUN1/2 and the outer nuclear envelope via a family proteins with a KASH domain (Nesprins) that then connect to the actin, intermediate filament and microtubule network.

Disorders linked to the nuclear membrane

Null or missense mutations in genes encoding LINC complex proteins (EMD, LMNA, SYNE1 and SYNE2) lead to a distinct muscular dystrophy syndrome with proximal weakness, joint contractures and cardiomyopathy termed Emery-Dreifuss Muscular Dystrophy (EMD)¹⁴⁶. EMD muscle pathology has both myopathic and dystrophic features. Ultrastructural features include altered myonuclear structure with nucleoplasmic extrusion¹⁴⁶. The pathophysiology of “nuclear envelopopathies” may be multifactorial. Loss or mutations in LINC complex proteins affects nuclear migration and myonuclear organization that are essential for a multinucleated cell such as skeletal muscle¹⁴⁷. Nuclear lamins anchor chromatin and disease mutations alter gene expression via altered epigenetic modifications such as methylation¹⁴⁹. A surprising consequence of LINC complex dysfunction is a loss of mechanical stiffness generally thought to be due to dysfunction at the sarcolemma¹⁵⁰. This finding emphasizes the necessary connection of the nuclear matrix to the extracellular matrix via the cytoskeletal proteins.

Impaired nucleocytoplasmic transport is seen in motor neuron disease and is associated with the cytosolic accumulation of RNA granules such as stress granules or protein inclusions¹⁵¹. In muscle, a rare form of LGMD, LGMDD2/1F, is due to dominant mutations in the nuclear transport receptor transportin-3 (TNPO3)¹⁵². TNPO3 is a cargo receptor for SR domain containing RNA binding proteins and is essential for HIV infection¹⁵³. Notably, cells from patients with LGMDD2 are immune to HIV infection¹⁵³.

Therapeutic approaches for disorders linked to myonuclei

Gene replacement strategies may be appropriate for diseases such as EMD associated with the loss of EMD. However other approaches focus on correcting the epigenetic dysregulation that occurs with LMNA mutations. For example, in mice homozygous for the H222P LMNA missense mutation associated with a recessive form of EMD, histone methylation and more specifically H3K4me1 was reduced at key regulatory regions¹⁴⁹. Correction of this by inhibiting an H3K4me1 demethylase (LSD1) was demonstrated to improve cardiac dysfunction in Lmna mutant mice. Using this same mouse model, inhibition of mTOR and subsequent activation of autophagy have been demonstrated to improve cardiac function and pathology¹⁵⁴. The improvement correlated with a reversal of metabolic deficits such as elevated lipolysis and reduced thermogenesis¹⁵⁵. These therapeutic approaches highlight the complicated pathophysiology seen in these disorders.

Heading: Defective Ca2+ handling

Introduction to excitation contraction coupling and the skeletal muscle triad

Skeletal muscle is electrically excitable. It is innervated by motor neurons that project from the spinal cord and synapse on each muscle fiber at the neuromuscular junction (NMJ). An electrical signal, initiated at NMJ following neuronal activation, travels transversely along the sarcolemma and subsequently down into the muscle via transverse tubules (T-tubules), which are invaginations of the sarcolemma (Figure 4). The T-Tubule interacts with the terminal sarcoplasmic reticulum (site of calcium storage) at a specialized junction call the triad. The triad facilitates calcium release from the SR and into the sarcoplasm, which promotes muscle contraction via relaxation of inhibition of actin-myosin cross bridging. The two main proteins of the triad are the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR or Cav1.1). Mutations in RYR1 and CACNA1S (which encodes a Cav1.1 subunit) are associated with a range of skeletal myopathies, including core myopathies, malignant hyperthermia susceptibility, and periodic paralysis⁹¹.The triad is composed as well of numerous proteins that support its formation, maintenance and function, and mutations in several of the genes encoding these proteins results in muscle disease^156,157.

Figure 4 |. The triad is a muscle-specific substructure that is critical for mediating excitation contraction coupling.

The triad represents the apposition of a Transverse (T)-tubule with two terminal cisternae of the sarcoplasmic reticulum (SR). While many types of proteins and regulatory structures located in or around the triad have been identified, the mechanisms of T-tubule biogenesis and triad formation are still incompletely understood. a | A muscle fiber is excited by the motor neuron at the neuromuscular junction, inducing membrane depolarization, which travels along the sarcolemma and into the t-tubule. The dihydropyridine receptor (DHPR) senses membrane depolarization, and undergoes a conformation change which activates the ryanodine receptor (RYR1), releasing Ca²⁺ from the SR into the sarcoplasm. Sarcoplasmic Ca²⁺ then binds to the troponin complex, releasing inhibition and initiating a muscle contraction. Several other proteins are critical for the formation and maintenance of the triad. MTM1 potentially regulates transport of muscle-specific proteins to the triad. BIN1 is involved in membrane remodelling, and in concert with caveole, initiates T tubule formation. DNM2 is speculated to function in parallel (and potentially opposed) to BIN1, via its membrane fission activity, to modulate T-tubule formation and maintenance. Two examples of mutations and their consequences on the triad: b | DNM2 hyperactivity leads to aberrant/premature membrane fission and abnormal t-tubule formation; b’ | RYR1 mutations lead to impaired calcium release and reduced muscle contraction.

RYR1 and RYR1 related myopathies

RYR1 encodes the skeletal muscle ryanodine receptor (RyR1), a massive homo-tetrameric calcium release channel located in the terminal sarcoplasmic reticulum¹⁵⁸. RyR1 is the protein that releases calcium from the SR to the sarcoplasm during ECC. Mutations in RYR1 cause a range of myopathies¹⁵⁹, which in total represent the commonest group of non-dystrophic pediatric muscle diseases, with prevalence estimates as high as 1:10000¹⁶⁰. Mutations can be broadly classified as resulting in either reduced or increased regulated calcium release¹⁵⁶. Reduced expression/function mutations are generally associated with static muscle weakness and cause myopathies including central core disease^161,162, mini core myopathy¹⁶³, congenital fibre type disproportion¹⁶⁴, and centronuclear myopathy¹⁶⁵. These myopathies historically have been named for findings on muscle biopsy, but the field is now evolving toward a “gene first” definition, encompassing all of these entities as RYR1 related myopathies (RYR1 RM)¹⁶⁰. Recessive mutations, most typically seen with minicore and centronuclear pathology, tend to result in both reduced function and expression, and are associated with more severe clinical phenotypes, including the need for ventilator and wheelchair supports^166,167. Dominant mutations that cause myopathy most typically are missense, reduce function, and result in central core pathology^166,167. Dominant/missense mutations that increase calcium release in response to stimuli predispose to dynamic conditions, particularly malignant hyperthermia^168,169, though can also include exertional rhabdomyolysis¹⁷⁰ and exertional heat illness¹⁷¹. About 30% of patients manifest both these dynamic “hyper-kinetic” conditions as well as static muscle weakness.

At present, there are no effective therapies for RYR1 RM. Aberrant oxidative stress has been identified in pre-clinical models and patient cells^172,173, associated with both gain and loss of function mutations, and is thus an attractive pathway for intervention. The anti-oxidant N-acetylcysteine has shown efficacy in zebrafish and mouse models^172,173, but unfortunately failed to meet the primary endpoint in a randomized, placebo controlled clinical trial¹⁷⁴. Anti-oxidants with increased muscle penetration are now being examined, as one caveat to the trial result was the fact that oxidative stress in treated patients was not significantly reduced with NAC. Another disease mechanism that is potentially therapeutically tractable relates to mutations that impact RyR1 binding to calstabin, impairment of which causes chronic calcium leak from the RyR1 channel. A class of compounds called Rycals stabilize this interaction and prevent calcium leak, as has been shown in pre-clinical models including ex vivo studies of patient muscle biopsies¹⁷⁵. Based on these promising results, Rycals are now being considered for testing in the clinical trial arena.

CACNA1S and CACNA1S related myopathies

Cav1.1, also called the dihydropyridine receptor (or DHPR) is an oligomeric protein complex composed of 5 subunits^176,177. Cav1.1 is an L-type calcium channel which functions as a voltage sensor during EC coupling. Membrane depolarization initiated at the NMJ travels down the T tubule until reaching the DHPR, which undergoes a conformation change in response that promotes opening of the RyR1 calcium channel. CACNA1S encodes the alpha 1 subunit of DHPR¹⁷⁸, which is the largest subunit and the one primarily responsible for interaction with, and regulation of, RyR1. CACNA1S is the only subunit mutated in skeletal myopathies; mutations are associated with a range of phenotypes including malignant hyperthermia¹⁷⁹, exertional heat illness¹⁸⁰, periodic paralysis¹⁸¹, and myopathy¹⁸². CACNA1S mutations account for approximately 5% of all cases of MH (as opposed to RYR1 mutations, which cause 70%). MH related mutations lie in the domain of CACNA1S that directly interacts with RyR1 and disrupts the negative regulatory function of CACNA1S on RyR1 calcium release. CACNA1S mutations are one of two main causes of hypokalemic periodic paralysis (the other being SCN4A), a condition of muscle inexcitability that manifests as prolonged episodes of flaccid muscle weakness that can last hours to days. Mutations resulting in periodic paralysis reside in the voltage sensing domain, with two alleles accounting for the majority of cases. Individual episodes of paralysis can be aborted with potassium supplementation and reduced by use of acetazolamide and avoidance of known triggers¹⁸³. However, a progressive myopathy with limb girdle weakness usually emerges in adulthood.

A congenital onset myopathy has recently been described associated with mutations in CACNA1S¹⁸². This myopathy resembles recessive RYR1 related myopathy and includes early onset, severe weakness, and involvement of the muscles of eye movement. Histopathology is variable, and may include central nuclei, core or core like lesions, myofibrillar disorganization, and fiber type disproportion, plus the unique feature of a lobular or “alveolar” appearance with oxidative stains. Both dominant and recessive mutations have been identified, with most missense mutations located in regions encoding cytoplasmic loops. Recessive patients have either two nonsense mutations or one missense and one nonsense, while dominant/de novo patients all have missense variants. Interestingly, the majority of patients studied, regardless of mutation type, have reduced CACNA1S protein levels, suggesting loss of expression/function as the main disease pathomechanism. Studies of intracellular calcium dynamics support this, as EC coupling is impaired in the mutations investigated.

STAC3 myopathy

A third essential component of the EC coupling apparatus is encoded by the STAC3 gene. STAC3 is a bridging protein that is required for triad formation (likely by chaperoning Cav1.1 to the triad) and for regulation of the Cav1.1/RyR1 interaction¹⁸⁴. Biallelic mutations in STAC3 were first identified in a rare muscle disorder called Native American Myopathy, a condition described in the Lumbee Native Americans of North Carolina and featuring facial weakness with prominent ptosis, extremity weakness and contractures, and MH susceptibility¹⁸⁵. All individuals with NAM are homozygous for a recurrent missense variant in STAC3. Additional non-Lumbee individuals with recessive STAC3 mutations have been identified with a similar phenotype that includes congenital onset weakness, facial involvement, short stature, joint contractures, and MH susceptibility¹⁸⁶. The same “NAM” mutation has been found in some, and additional variants have also been identified.

Centronuclear myopathy due to mutations in MTM1, DNM2, BIN1, and SPEG

Centronuclear myopathy is a congenital onset muscle disease united by features on muscle biopsy including >25% of fibers with central nuclei, myofiber hypotrophy, and organelle disorganization (as seen with oxidative stains). The genes underlying CNM encode proteins involved in membrane traffic¹⁸⁷. A hallmark feature of CNM is disturbance of the formation and/or maintenance of the structure of the triad^188,189, the result of which is impaired EC coupling and severe muscle weakness.

Mutations in the X-linked gene MTM1 were the first described cause of CNM¹⁹⁰. MTM1 encodes a ubiquitously expressed phosphoinositide phosphatase that is a component of the endolysosome and that is a key regulator of endosomal membrane sorting. Loss of expression/function mutations result in X-linked myotubular myopathy, a devastating myopathy affecting primarily males that is associated with profound weakness, ventilator and feeding tube dependence, and early death in most individuals^191–193. The exact function(s) of MTM1 in skeletal muscle, and the reasons why mutations impair triad structure and function, are incompletely understood. MTM1 has been implicated in stabilizing myofiber organization via interaction with desmin and in regulating intermediate filament turnover via interaction with the ubiquitination machinery^194,195. How these molecular interactions may impact the triad is unclear. Despite this lack of knowledge, several therapies have been identified that ameliorate pathology and clinical phenotypes of animal models of the disease¹⁹⁶. These include gene replacement therapy¹⁹⁷, phosphoinositide rebalancing via PIK3C2B inhibition¹⁹⁸, and DNM2 reduction (either with antisense oligonucleotide or with tamoxifen)^199,200. Building from these targets and robust natural history clinical data, the first clinical trials for XLMTM have been initiated. Preliminary reporting of gene therapy with AAV8-MTM1 are encouraging, though important safety concerns have also emerged^201,202.

DNM2 encodes a ubiquitously expressed large GTPase that functions as a molecular scissor to promote membrane fission²⁰³. DNM2 protein is implicated in myriad cellular functions, including clathrin-mediated endocytosis and cytokinesis. Dominant/de novo mutations in DNM2 cause a form of autosomal centronuclear myopathy²⁰⁴, similar in characteristics to XLMTM but in general milder in severity. Mutations are thought to result in hyper-functionality of DNM2, either by releasing auto-inhibition or prolonging protein stability²⁰⁵. As with MTM1, the exact mechanism by which DNM2 mutations impact the triad is unclear, though data supports a theory where-by mutations lead to premature or inappropriate fission of the maturing T tubule. Interestingly, increased levels of DNM2 are found in muscle from XLMTM and from BIN1 related CNM²⁰⁶, and overexpression of DNM2 in mice leads to a CNM like phenotype²⁰⁷. Therefore, DNM2 overexpression or hyper-function may be the key aspect that drives CNM pathology. This concept is the basis for the therapeutic strategy of lowering DNM2 levels in the various genetic subtypes of CNM²⁰⁸, and an antisense oligonucleotide based approach has shown efficacy in mouse models of MTM1, DNM2 and BIN1 CNM^200,206,209.

BIN1, or amphiphysin 2, is a membrane deforming protein critical for the formation of the T tubule²¹⁰. It also interacts directly with DNM2²¹¹. Recessive mutations, associated primarily with loss of expression, cause CNM²¹¹. SPEG encodes a large protein kinase in the obscurin and myosin light chain kinase family. Recessive mutations cause a lethal subtype of CNM also associated with cardiomyopathy²¹². SPEG interacts directly with MTM1, and loss of SPEG secondarily impacts MTM1 expression²¹². SPEG also likely phosphorylates key target proteins at the triad (as demonstrated in cardiac myocytes, and including SERCA and potentially RyR1)^213,214. Its function, and the consequences of mutation, thus may be a combination of interplay with MTM1 and its role as protein kinase.

SELENON related myopathies

The SELENON gene encodes a cytoplasmic protein (SePN) in the selenocysteine family of proteins²¹⁵. SEPN appears to function as a critical regulator of cellular stress. Recessive mutations in SELENON are described in patients with a consistent clinical picture (severe and progressive axial weakness, rigid spine with scoliosis, and disproportionately mild extremity weakness) and a range of histotypes (minicore myopathy, congenital fiber type disproportion, Mallory body myopathy, and rigid spine muscular dystrophy)^215–219. There is likely an important interplay between SEPN, calcium homeostasis, and regulated calcium release from the triad. As with RYR1 mutations, aberrant oxidative stress has been uncovered in models of SELENON related myopathy, and anti-oxidants such as N-acetylcysteine ameliorate these changes²²⁰. Clinical translation of this strategy is in progress.

Store operated calcium entry (SOCE)

While not essential for EC coupling, store operated calcium entry (or SOCE) is critical for maintaining calcium homeostasis in the myofiber²²¹. The main components of the SOCE machinery are the calcium channel ORAI1 and its activator STIM1. Depletion of calcium from the sarcoplasmic reticulum induces a conformational change in STIM1, which, in turn, activates ORAI1, leading to extracellular calcium entry into the sarcoplasm. Mutations in both ORAI1 and STIM cause disease that impacts multiple organ systems²²². Dominant, gain of function mutations lead to excessive calcium entry and are most relevant to skeletal muscle, as they are associated with tubular aggregate myopathy and Storkmorken Syndrome²²³.

Impaired sarcomere remodeling due to impaired Ca²⁺ handling

Healthy muscle can remodel and change its contractile and metabolic properties to meet the physiological demands placed upon it. This process involves Ca²⁺ mediated signaling that alters gene expression of slow/oxidative and fast/glycolytic (FG) gene expression programs. During muscle deconditioning (i.e. bed rest), muscle shifts to a more FG phenotype, while during muscle conditioning, it shifts towards the SO type. Studies in the mouse model of LGMD R1 (Capn3 knock out)²²⁴ have revealed a role for calpain 3 (Capn3) in Ca²⁺ handling, muscle remodeling and adaptation^224–226. Capn3 is the muscle specific member of a family of cysteine proteases, collectively referred to as calpains²²⁷. Like other Capns, the active site aligns through changes in Capn3’s secondary structure, induced by Ca²⁺-calmodulin binding and autolytic cleavages^228,229. Capn3 homodimerizes through its C terminal PEF domains²³⁰ and anchors on the giant protein titin^231,232. LGMD R1 mutations have been shown to interfere with either Capn3 activity, anchorage to titin or homodimerization^233,234. Capn3 proteolytically cleaves titin as well as other sarcomeric proteins such as filamin C (FLNC)²³⁵ and myosin light chain 2 (MLC2)²³⁶ and these cleavages target sarcomeric proteins to the proteasome.²³⁷

Capn3 at the triad

In addition to Capn3’s anchorage on the N2 line of titin (the site where the T tubule enters the myofibril), Capn3 also binds to RyR1 and stabilizes the triad protein complex²³⁸. Reductions in RyR1 levels²²⁶ are observed in both Capn3 knock out and LGMD R1 patient biopsies and reduced Ca²⁺ transients are observed in Capn3 KO mice²³⁹. Other proteins that are known to be enriched at the triad, such as aldolase²³⁸ and calcium calmodulin kinase (CaMKII),²²⁶ are also greatly reduced in Capn3 knock out mice. Thus, in addition to its proteolytic role, Capn3 plays a structural role in maintenance of triad complex integrity.

Loss of triad integrity leads to severe reductions in CaMKII levels and activation^225,226. Because CaMKII promotes the slow oxidative gene expression program, loss of Capn3/CaMKII leads to impaired expression of slow/oxidative genes and failed skeletal muscle remodelling. The phenotypic features of mitochondrial abnormalities^241,242 growth failure²²⁴, sarcomere disorganization^224,237, and abnormal fat metabolism²²⁵ in Capn3 deficient muscles can be explained by reductions in the slow-oxidative gene expression program.

The identification of CAPN3 as the defective gene in LGMD R1 was the first discovery of a gene mutation linked to a LGMD^243,244. Pathogenic mutations occur along the entire molecule and approximately 60% are missense mutations. Some missense mutations impact proteolytic activity while others likely impact either secondary structure, titin binding or calmodulin binding^229,234,245. Genotype/phenotype correlations have been hindered by the fact that most patients are compound heterozygotes. Additional studies are needed to unravel the relationship between Capn3, titin and CaMKII signalling and the impact of these biochemical changes on disease features.

Skeletal muscle channelopathies

Skeletal muscle is an electrically active tissue, and charge balance between the sarcoplasm, the SR, and the extracellular milieu is critical for maintenance of muscle tone and to ensure proper cycling between the relaxed and contracted state. This balance is regulated by several important ion channels, including the sodium channel SCN4A and the chloride conductor CLCN1. Mutations in these genes, as well as several others, cause a group of muscle disorders collectively referred to a skeletal muscle channelopathies. Phenotypes include periodic paralysis (mentioned above in reference to CACNA1S) and myotonia (prolonged muscle stiffness), as well as some forms of congenital myopathy. An in-depth examination of these important ion channels and the disorders associated with them is presented in these reviews^246,247

Heading: Defects of membrane repair

Introduction to skeletal muscle membrane repair and lipid trafficking

The muscle sarcolemma experiences high forces during muscle contraction, and these forces can lead to small sarcolemmal ruptures. These membrane tears cause increased local calcium entry and a change in phosphatidyl serine from the inner leaflet to the outer leaflet of the membrane. Annexin proteins A6, A1 and A2 sense the high calcium and phospholipid breakdown and they traffic to the injury site where they oligomerize and form a “repair cap”^248,249. Annexin A6 spreads more broadly across the repair cap than the other annexins²⁵⁰ and it likely contributes to the membrane curvature needed for the cap to form²⁵¹. The increase in local calcium also activates calcium-dependent proteases (calpains I and II, but not calpain 3²⁵²), which have been shown to proteolytically cleave dysferlin, and release a C terminal fragment) that has been referred to as “mini-dysferlin”¹⁰. Dysferlin may be a synaptotagmin-like molecule²⁵³, functioning to recruit lysosomal vesicles to the injury site²⁵⁰, likely through a process that is similar to mechanisms of neurotransmitter release²⁵⁴. Trim72 (AKA MG53) participates in vesicle trafficking to the injury site, although it is not clear if mini-dysferlin and Trim72 work cooperatively in the repair process.²⁵⁵ Membrane lipids are also recruited from the membrane, lateral to the injury site.²⁴⁸ At the site of repair, mini-dysferlin concentrates at the base or “shoulder” of the repair cap along with Trim72²⁴⁸, EHD proteins, and BIN1²⁴⁸. Annexins, Trim72 and dysferlin have all been shown to associate with phosphatidyl serine, which may be part of the repair signaling.^255,256 After the membrane is repaired, the repair cap and excess Ca²⁺ are shed via exocytosis mediated by Trim72.^257,248 The repair process involves microtubules, actin filaments and dynamin^{248,258–260}

Disorders linked to defective membrane repair

Loss-of-function mutations in the DYSF gene, which encodes dysferlin, underlie LGMD2B (LGMD R2) or the allelic distal myopathy called Myoshi Myopathy^261,262. Studies in the Dysf knock out mouse have revealed an essential role for dysferlin in skeletal muscle membrane repair²⁶³. Defective membrane repair may also underlie LGMD2L due to mutations in ANO5²⁶⁴, which encodes Anoctomin 5 protein. Ano5 knock-out mice show impaired membrane repair, although it is not clear whether Anoctomin 5 directly participates in the repair process.

Treatment of membrane repair disorders

Gene replacement therapies for LGMD2B/Myoshi myopathy are in pre-clinical development, but the large size of the DYSF gene (6.5 kb) prevents its packaging into a single AAV vector. Thus, strategies that utilize two, overlapping vectors have been tested in murine models and have shown some success²⁶⁵. Gene replacement therapies are also in development for LGMD2I (FKRP) and LGMD2L (ANO5).

Dysferlin deficiency is associated with high levels of intramuscular inflammation, consisting mainly of T cells and macrophages^266–270. Given the inflammatory state of dysferlin-deficient muscles, one would expect steroids (an effective therapy for DMD) to be beneficial for LGMD2B, but this is not the case with daily steroid treatment. Weekly treatment may prove to be more beneficial, because the positive effects of steroids on membrane repair outweigh the negative effects on muscle wasting and metabolic reprogramming observed with daily steroids^271,272. On the other hand, human biopsies have shown high levels of the complement-associated membrane attack complex (MAC) on the surface of skeletal muscle fibers of LGMD2B pateints²⁷³, and studies in the murine model of LGMD2B have demonstrated benefit from interference with the complement pathway²⁷⁴. A small study that used Rituximab (B cell depletion) led to increased muscle strength in two patients who were tested²⁷⁵. Systemic administration of recombinant A6 protein showed benefit in reducing membrane injury in the mouse model of LGMD2C (SGCG knock out mice)²⁴⁸.

Heading: Failure of protein quality control

Introduction to cellular systems that regulate protein quality in muscle

All cells maintain a balance between protein synthesis and protein degradation. However, skeletal muscle in particular utilizes this balance to increase muscle mass via enhanced protein synthesis (hypertrophy) or to decrease muscle mass via enhanced proteolysis (atrophy) under conditions of exercise and inactivity. Moreover, the physiologic stress and strain placed on myofibers and long-lived structural proteins within myofibrils results in their repeated unfolding and refolding that is necessary to prevent protein degradation or aggregation. Additionally, skeletal muscle is exquisitely sensitive to changes in organismal nutrient state and serves as the largest reservoir of free amino acids under conditions of starvation. The protein degradation in skeletal muscle that occurs with disuse, denervation, anorexia or chronic illness is mediated through two principal proteolytic pathways, the ubiquitin proteasome system (UPS) and autophago-lysosomal pathway (ALP).

Myopathies associated with ALP dysfunction

Autophagy and more specifically macroautophagy is the selective sequestration and subsequent engulfment of proteins, organelles and cytoplasm by an expanding phagophore that becomes an autophagosome. Cargo filled autophagosomes are then trafficked to the late endosome/lysosome enabling proteolysis within an acidic milieu. Mutations in both autophagic and lysosomal proteins lead to myopathies with distinctive pathologic features that include autophagic and lysosomal vacuoles. Specifically, inactivating mutations in proteins necessary for autophagosome lysosome fusion such as EPG5 and LAMP2 cause Vici Syndrome and Danon’s disease respectively^276,277. These myopathies have the characteristic accumulation of autophagic and lysosomal vacuoles as evidenced by the accumulation of autophagic and lysosomal proteins such as SQSTM1, LC3 and acid phosphatase with associated multisystem involvement including both cardiac and CNS manifestations^276,277. Inactivating mutations in proteins necessary for normal lysosomal function can also lead to a vacuolar myopathy. For example, loss of the acidic hydrolase, GAA or a reduction in VMA21, the chaperone necessary for the assembly of the vacuolar-type ATPase both lead to lysosomes with reduced degradative capacity^278,279. The pathology in these myopathies termed Pompe’s disease and X-linked myopathy with excessive autophagy (XMEA) is distinctive with the sarcoplasmic accumulation of vacuoles surrounded by sarcolemmal proteins such dystrophin and caveolin-3²⁸⁰.

Myopathies associated with UPS dysfunction

Under conditions of muscle atrophy, the UPS is activated leading to the rapid degradation of myofibrillar proteins and reduction in muscle mass. Proteins are selectively targeted and degraded via the UPS. The selectivity of this process is dictated by a large family of ubiquitin ligases or E3 ligases. During muscle atrophy expression of the E3 ligases muscle RING-finger 1 (MuRF1) and Atrogin1/MAFbx ubiquitinate thick filament proteins such as myosin²⁸¹. The E3 ligase Trim32 was originally thought to ubiquitinate thin filament proteins²⁸²; however, its role is more likely to be control of muscle stem cell^283,284 possibly through regulation of Piasy²⁸⁵, an E3 SUMO ligase or NDRG2²⁸⁶. Consistent with this critical role in muscle proteostasis, homozygous loss of function mutations in MURF1 lead to myopathies with vacuoles and sarcoplasmic inclusions. Mutations in TRIM32 underlie LGMD2H, sarcotubular myopathy and the allelic disorder Bardet Beadle Syndrome. The substrate specificity of some E3 ligases require adaptor proteins necessary for efficient ubiquitination. Loss of function mutations in three related Kelch proteins, KLHL40, KLHL41 and KBTBD13 lead to nemaline rod myopathies suggesting their importance in the proteasomal degradation of thin filament proteins²⁸⁷.

Myopathies associated with chaperone dysfunction.

Chaperones are proteins necessary for protein quality control or “proteostasis.” In lower organisms, this group of proteins is critical for cell survival following environmental insults that destabilize proteins such as heat shock and are thus termed heat shock proteins (HSPs). HSPs are a multi-tiered network of proteins that include HSP70s/HSPAs, HSP40s/DNAJs, HSP20s/HSPBs and BAG family proteins. A disruption in the HSP network leads to the accumulation of misfolded and aggregated proteins that can further disrupt protein degradation pathways such as autophagy. Dominant missense mutations in the co-chaperones DNAJB6 (LGMDD1) or BAG3 (MFM6) lead to myopathies with Z-disc disorganization and desmin inclusions^288–290. The dominant effect may relate to the mutant proteins’ avidity and sequestration of other HSP proteins (e.g. HSPA1) in aggregates. Mutations in the small HSPs, HSPB5 and HSPB8 also lead to myopathies with prominent myofibrillar disorganization and rimmed vacuole^291,292. Finally, disruption of chaperone proteins within the secretory pathway result in myopathies with aggregates and vacuoles. For example, loss of function mutations in Sil1 (Marinesco-sjogren syndrome) a co-chaperone for HSPB5/Bip lead to a congenital myopathy with rimmed vacuoles^293,294. Notably therapies aimed at restoring protein homeostasis such as HSP activators may be effective in chaperonopathies.

- Conclusions and perspectives:

Mutations that perturb normal processes in skeletal muscle result in myopathies or muscular dystrophies. Over the past three decades, hundreds of such mutations have been identified. In-depth study of these disease-causing proteins has led to an immense amount of new information regarding the cellular processes necessary to maintain healthy muscle. Understanding the normal function of these proteins has provided valuable insights on why mutations cause disease and will be necessary for development of efficacious therapies. The availability of the underlying genetic causes of these diseases provides a facile path to develop therapies targeting the underlying cause of disease such as gene replacement, gene editing and antisense oligonucleotide-based exon skipping.

Figure 5 |. Skeletal Muscle Membrane Repair.

Skeletal muscle sustains small tears from normal muscle use, which are quickly repaired by cell repair machinery. a | Following a tear, calcium flows down its concentration gradient from outside the cell to inside. Cholesterol is oxidized and phosphatidyl serine changes its conformation from intracellular-facing to extracellular facing. b | Calcium, oxidized cholesterol and phospholipids activate the cellular repair machinery. Annexin proteins bind to calcium and phosphatidyl serine and form a cap that seals the are of the tear.

Figure 6 |. Schematic of a muscle cell and the proteins linked to protein turnover and quality control.

b | Skeletal muscle mass is maintained by the balance of protein synthesis and protein degradation. b | Disruptions in chaperone surveillance, the ubiquitin proteasome system and the autophago-lysosomal pathway lead to pathologic changes in skeletal muscle that include myofibrillar disruption, inclusion bodies, protein aggregates and vacuolation.