Extralysosomal Protein Degradation in Myofibrillar Myopathies

Abstract

Myofibrillar myopathies (MFMs) are a group of heterogeneous muscle disorders morphologically defined by the presence of foci of dissolution of the myofibrils, accumulation of the products of myofibrillar degradation and ectopic expression of multiple proteins. MFMs represent the paradigm of conformational protein diseases of skeletal and cardiac muscles. Protein aggregation in MFMs is now considered to be the result of a failure of the extralysosomal proteolytic degradation system. Several factors including mutant proteins, aggresome formation and oxidative stress may compromise the ubiquitin–proteasome system, promoting the accumulation of potentially toxic protein aggregates within muscle cells.

INTRODUCTION

Proper protein folding is an essential post‐translation process for correct protein function. Newly synthesized proteins must fold into their correct three‐dimensional structure and maintain these native states throughout their life. Several factors, including gene mutations, post‐translational modifications, oxidative stress, among others, may induce an abnormal change in protein conformation. Protein quality control systems are therefore necessary to help cells to cope with these conformationally abnormal proteins. Molecular chaperones represent the first line of defense against protein misfolding and aggregation, and allow protein repair or refolding. When chaperones fail to repair, they send the altered protein to be degraded by the ubiquitin–proteasome system (UPS), or by the hsp 70 chaperone‐mediated autophagy. If the activity of these two systems is impaired, macroautophagy is the only system that is able to remove those toxic protein complexes. When even autophagy is not able to remove the altered proteins, these aggregates often entrap other still‐functional proteins inside the cell 16, 23, 32, 36, 77.

Intracellular accumulation of altered or misfolded proteins is the basis of many neurodegenerative disorders generically known as protein conformational disorders 8, 15, 16, 55, 67, and is now also considered to play an important pathogenetic role in an expanding group of muscle disorders collectively called protein aggregate myopathies (PAMs) 28, 29. Myofibrillar myopathies (MFMs), representing the largest group of PAMs, are a group of heterogeneous muscle diseases having as a common feature the presence of focal dissolution of the myofibrils, accumulation of the products of myofibrillar degradation and ectopic expression of multiple proteins 14, 44, 64. MFMs have been associated with mutations in several protein components of the Z‐disk including desmin 30, 31, 43, αB‐crystallin (73), myotilin 45, 62, ZASP (63), filamin C (74) and BAG3 (65). Protein aggregates in MFMs are composed of a wide variety of proteins 14, 17, 44, 64. Some of them are normal constituents of the myofibrils; however, many nonspecific muscle proteins are aberrantly expressed in muscle fibers as well. Protein aggregation in MFMs is now considered to be the result of a failure of proteolytic degradation systems of cells and not of increased synthesis 17, 18, 28, 29. Several factors, including mutant proteins, oxidative stress and aggresome formation, among others, are thought to compromise the UPS, and maybe autophagy as well, facilitating the accumulation of large potentially toxic protein aggregates that ultimately result in muscle fiber degeneration. In this review, we summarize the mechanisms contributing to defective extralysosomal protein degradation in MFMs.

Protein composition of aggregates in MFMs

As stated earlier, intracytoplasmic accumulation of insoluble protein aggregates in muscle cells constitutes a major morphological hallmark of MFMs. Besides the respective mutant protein in each subgroup of MFMs (ie, desmin in desminopathies, αB‐crystallin in αB‐crystallinopathies, myotilin in myotilinopathies), protein aggregates in MFM are enriched with a wide variety of proteins. These include ubiquitin, phosphorylated neurofilaments, tau, β‐amyloid, plectin, gelsolin, syncoilin, synemin, caveolin, dysferlin, heat shock proteins, α1‐antichymotrypsin and many others, suggesting that mutant proteins facilitate the entrapment of other proteins within the muscle cells (14, 44, 64 reviewed in 17) (1, 2).

Figure 1.

Muscle biopsy from a patient carrying a Pro419Ser desmin mutation. Increased variation in muscle fiber diameters and mild endomysial fibrosis, modified Gomori's trichrome stain (A). Abnormal fiber areas show accumulation of desmin (B), αB‐crystallin (C), phosphorylated neurofilaments as revealed with BF‐10 (D) and SMI‐31 (E) antibodies. Nonserial section showing aberrant synaptophysin expression (F). A–F ×200.

Figure 2.

Muscle biopsy with increased variation in muscle fiber diameters and vacuoles within few muscle fibers, hematoxylin–eosin (A), from a patient with a Ser55Phe myotilin mutation showing prominent protein aggregates containing myotilin (B), filamin C (C), phosphorylated neurofilaments (D), UCHL‐1 (E) and α‐internexin (F). A, E and F ×200; B–D ×400.

Genes coding for most of the proteins found accumulated in myofibers in MFMs are not up‐regulated compared to controls 47, 52, thus suggesting that protein aggregates are not the result of increased protein synthesis. Rather, they are the result of post‐translational modifications of proteins that render them resistant to the protein degradation systems of muscle cells.

The role of the UPS

The UPS is responsible for the selective degradation or recycling of short‐lived intracellular and nuclear proteins 23, 27, 37. Misfolded proteins or unassembled subunits of larger protein complexes and retro‐translocated proteins from the endoplasmic reticulum are also subject to proteasomal degradation. Targeted proteins must become conjugated to a polyubiquitin chain to be recognized by the proteasome. Ubiquitin conjugation is a highly ordered process. In the first step, a ubiquitin activation enzyme E1 activates and transfers ubiquitin to one substrate‐dependent ubiquitin‐conjugating enzyme, E2. Ubiquitin is then transferred to a member of the ubiquitin ligase family, E3. This is followed by poly‐ubiquitination of the substrate, produced via a series of isopeptide linkages between a lysine residue of the attached ubiquitin and the carboxyl‐terminal glycine of the next ubiquitin molecule 23, 27, 37, 70. In addition to ubiquitylating enzymes, additional factors including deubiquitylating enzymes, shuttling factors and chaperones are involved in presenting substrates to the proteasome for degradation (70).

The 20S proteasome is organized into a structure resembling a hollow cylinder composed of four stacked rings, each composed of seven subunits. The catalytic β‐subunits are located in the inner two rings, while the gated α‐subunits are located in the outer two rings. The 20S proteasome can associate, in the presence of ATP, with two caps or 19S complexes that bind to the outer rings of the 20S complex, thus forming the 26S proteasome complex. The 20S proteasome has three main peptidase activities: chymotrypsin‐like, trypsin‐like and peptidylglutamyl peptide hydrolyzing activities 27, 70.

The 20S proteasome can also bind to the PA28α/β activator to form the PA28‐proteasome complex, which appears to increase antigen processing. In the presence of certain stimuli, the β‐subunits of the 20S proteasome are replaced by homologous proteins, called LMP2, LMP7 and MECL1, forming the immunoproteasome. Hybrid proteasomes formed by 19S, 20S and PA28 are involved in the immune response 27, 32.

Protein aggregates in MFM are often ubiquitinated, suggesting involvement of the UPS. Yet, several proteasome subunits are abnormally expressed in muscle fibers in MFM as revealed by immunohistochemistry and Western blotting (18). Subunits of the 20S, 19S and PA28 colocalize with ubiquitin and other proteins, including desmin, αB‐crystallin, gelsolin and phospho‐tau, in abnormal protein aggregates in myotilinopathies (Figure 3). Moreover, fibers with protein aggregates express LMP2, LMP7 and MECL1(18). This is accompanied by preserved or even increased proteolytic activity in vitro in myotilinopathies (18).

Figure 3.

Colocalization of ubiquitin (A), αB‐crystallin (B), desmin (C) and 20S (D), 19S (E) and PA28 (F) components of the proteasome in muscle fibers in myotilinopathy. A–F ×200.

Studies in heart muscle from a transgenic mouse model of desminopathy have also demonstrated over‐expression of the 20S subunit and down‐regulation of the 19S subunit of the proteasome together with increased peptidase activity 39, 40.

These observations strongly suggest that abnormalities of the UPS in MFMs are not the result of defective peptidase activity per se, but rather respond to a failure in the entry of large ubiquitinated protein inclusions into the narrow barrel of the proteasome's proteolytic core.

Abnormal proteasomal expression and activity do not fully explain why several proteins cluster and aggregate into abnormal protein deposits in MFM. Studies in transgenic mice and transfected cell lines have demostrated that mutation of target proteins facilitates protein aggregation 3, 4, 5, 24, 25, 42, 43, 68, 73, 75, 76. Yet, additional complementary proteins may be involved in this process.

UCHL1 and other neural proteins in aggregates: role of neuron‐restrictive silencer factor

Ubiquitin carboxy‐terminal hydrolases (UCHLs) are enzymes involved in the hydrolysis of polyubiquitin chains to increase the availability of free monomeric ubiquitin molecules facilitating protein degradation (41). UCHL1 is highly expressed in brain and testis, but not in muscle. UCHL1 is present in inclusions associated with several neurodegenerative diseases, and it is considered to play a role in inclusion formation in Parkinson's disease 1, 41, 66. It has been recently demonstrated that mRNA and protein levels of UCHL1 are increased in myotilinopathies (6). The reasons for this phenomenon are unknown, but recent studies have shown that abnormal expression of certain transcription factors may be causative. NRSF/REST is a neuronal gene repressor in non‐neuronal cells 11, 58. Several studies have shown that synaptophysin and SNAP25 are NRSF/REST target genes (7). UCHL1 and α‐internexin are also regulated by REST (6). Interestingly, mRNA and protein NRSF/REST levels are reduced in myotilinopathy muscles when compared with control muscles. This is accompanied by increased aberrant expression levels not only of UCHL1, but also of other neuronal proteins such as SNAP25, synaptophysin and α‐internexin in skeletal muscles in myotilinopathies (6) (Figure 2E,F).

Mutant ubiquitin: a marker of proteasomal dysfunction

Mutant ubiquitin (UBB⁺¹) is an aberrant, potentially toxic form of ubiquitin generated by a non‐DNA‐encoded dinucleotide deletion occurring within UBB mRNA (72). The resulting protein has a modified C‐terminus, and is unable to ubiquitinate other protein substrates. UBB⁺¹ is ubiquitinated itself, and while at low expression levels it can be degraded by the proteasome, at high levels, it can inhibit the proteasome pathway 15, 38. Yet, the accumulation of UBB⁺¹ is considered a specific marker of proteasomal dysfunction in certain neurodegenerative disorders such as Alzheimer's disease, Down syndrome and other tauopathies 20, 72. Furthermore, UBB⁺¹ is expressed in muscle fibers in inclusion body myositis (21).

Aberrant expression of mutant ubiquitin UBB⁺¹ has been observed in myotilinopathies and desminopathies (46). UBB⁺¹ colocalizes with myotilin and desmin in both disorders as revealed in double‐labeling immunofluorescence and confocal microscopy studies. These observations suggest that the presence of UBB⁺¹ in muscle fibers in myotilinopathies and desminopathies is an additional factor contributing to dysfunction of the UPS in MFM.

p62

Sequestosome 1/p62 is a 62 kDa protein encoded by an immediate early response gene activated by a variety of extracellular signals (26). p62 participates actively in the formation of protein aggregates by recruiting poly‐ubiquitinated proteins through its UBA domain 60, 61. It has been proposed that p62 may promote aggregation and sequestration of abnormal proteins as inert but reversible inclusion bodies (48). Moreover, p62 binds specifically to protein aggregates, or to misfolded and ubiquitinated proteins in several human diseases, including neuronal and glial ubiquitinated inclusions in Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies and multiple system atrophy (78). It has recently been shown that p62 is also involved in the delivery of polyubiquitylated proteins for autophagy degradation (49).

p62 has been found colocalizing with myotilin in muscle fibers in myotilinopathies, and to a much lesser extent in desminopathies, as revealed in sections double stained for p62 and myotilin (46). The differing amounts of p62 in myotilinopathies and desminopathies may be related to the different degrees of compaction of myofibrillary inclusions in the two disorders, as well as to the less common formation of inclusion bodies in desminopathies.

Clusterin and the aggresome in MFM

Aggresomes are microtubule‐based inclusion bodies composed of aggregates of misfolded proteins near the centrosome. They are formed in response to cellular overload of abnormally folded proteins after the unfolded protein response and the UPS become overwhelmed and before autophagy is engaged. Aggregated proteins translocate to the aggresome by active transport (36). γ‐tubulin is thought to mediate the link between microtubules and the centrosome, and to function as a regulator of the microtubule‐organizing center.

Protein inclusions in MFM share features with aggresome structures as indicated by double‐labeling immunofluorescence and confocal microscopy. γ‐tubulin, which is considered a marker for aggresomes, colocalizes with clusterin, αB‐crystallin and myotilin in individual muscle fibers in MFMs (19) (Figure 4). Moreover, γ‐tubulin protein expression levels, as revealed by Western blotting, are increased in MFMs (19). Furthermore, the Arg120Gly mutation in the αB‐crystallin gene, which causes cardiomyopathy, forms aggresomes in vitro (9) and it is accompanied by aggresome‐related dense bodies containing desmin and αB‐crystallin in transgenic mice (56).

Figure 4.

Double‐labeling immunofluorescence and confocal microscopy to γ‐tubulin (green, A, D) and myotilin (red, B, E) in myotilinopathy. γ‐tubulin partially colocalizes with myotilin (merge, C, F). One section of the same case stained only with the secondary antibodies is used as a negative control (G, H). Nuclei are visualized with TO‐PRO‐3‐iodide (blue).

Another component of myofibrillary inclusions in MFMs is clusterin (apolipoprotein J, SP40), a sialoglycoprotein with a nearly ubiquitous tissue distribution (35). Clusterin shares functional homology with the small heat shock proteins. Clusterin participates in multiple physiological processes and has been associated in several pathological conditions, including conformational protein disorders affecting the central nervous system such as Alzheimer's disease, Parkinson's disease and prion diseases 10, 22, 54, 57. It has been proposed that clusterin gene expression is increased in response to altered protein homeostasis and oxidative stress injury (71).

Clusterin immunoreactivity is present in association with abnormal protein deposits in MFMs, as revealed by single‐ and double‐labeling immunofluorescence and confocal microscopy to clusterin and myotilin, αB‐crystallin and ubiquitin, thus indicating clusterin association with abnormal protein aggregates. Furthermore, Western blots have shown several clusterin‐immunoreactive bands between 34 and 80 kDa, probably corresponding to heterodimers and aggregates with other proteins in control and MFM cases (19).

Oxidative and nitrosative stress in MFMs

It is well known that oxidative and nitrosative damage promotes protein misfolding and aggregation. Oxidative stress results from an imbalance between the generation of reactive oxygen species and the level of antioxidant responses. Skeletal muscles are particularly prone to accumulating oxidative damage to DNA, lipids and proteins over time 2, 50, 69. Oxidized proteins are degraded by the UPS 12, 13, 51. However, proteasome activity drastically declines in cells containing large amounts of aggregated or oxidized proteins, and thereby facilitates the accumulation of abnormal proteins through covalent cross‐linking reactions and increased surface hydrophobicity 12, 13.

Recent studies have demonstrated increased levels of the glycoxidation markers AGE, CML and CEL, as well as of the lipoxidation markers MDAL and HNE, in muscle samples in myotilinopathies and desminopathies as revealed by Western blotting, immunohistochemistry and double‐labeling immunofluorescence and confocal microscopy (34). Furthermore, increased levels of nitrotyrosine occur in myotilinopathies and desminopathies when compared with controls (34). By means of immunohistochemistry, glycoxidation markers, particularly AGE and AGE receptor (RAGE), are seen to be aberrantly expressed in muscle fibers containing protein aggregates in myotilinopathies, and to a lesser extent in desminopathies. Interestingly, AGE colocalizes with myotilin, as well as with ubiquitin and p62, in abnormal fibers, thus providing a link between oxidative damage and abnormal protein aggregation in MFMs (Figure 5). In addition, neuronal, inducible and endothelial nitric oxide synthases (nNOS, iNOS and eNOS), as well as SOD2 are over‐expressed in abnormal muscle fibers in myotilinopathies and desminopathies. Detection of oxidized and nitrated proteins has been performed by means of bidimensional gel electrophoresis, in‐gel digestion and mass spectrometry in MFMs (33). As a result, desmin has been identified as a major target of oxidation and nitration in desminopathies and myotilinopathies. Additionaly, oxidized and nitrated mutant desmin has been identified in samples from desminopathies, whereas oxidized pyruvate kinase muscle splice from M1 has been found in myotilinopathies (33). Together, these observations strongly suggest that oxidative and nitrosative damage plays an important role in the pathogenesis of MFMs.

Figure 5.

Myofibrillary inclusions in myotilinopathy (Ser60Cys) showing p62 (A) colocalizing with MDAL (B), RAGE (C), nNOS (D), eNOS (E) and iNOS (F). A–F ×200.

The causes of oxidative stress leading to oxidative damage in MFMs are not fully known, but several concomitant factors may contribute to the progression of oxidative and nitrosative injuries in MFMs. Increased numbers of mitochondria in the vicinity of abnormal protein aggregates and isolated changes in individual mitochondria morphology are commonly seen in muscle biopsies from patients suffering from MFM. Low mitochondria respiratory chain complex I activity has also been reported in desminopathy 53, 59. Redox proteomic studies have shown that, in addition to desmin, the oxidation of mitochondrial enzyme pyruvate kinase is a putative component of impaired mitochondrial function, at least in some cases of myotilinopathy (33).

CONCLUDING COMMENTS

Proteinaceous inclusions in muscle cells constitute the morphological hallmark of MFMs. Identification of a number of proteins within the protein aggregates has led to the identification of causative genes, and has helped to better understand the mechanisms governing aggregation of these proteins. Mutant proteins, aggresome formation, p62, UBB¹⁺, oxidative and nitrosative damage, among other factors, may block the UPS resulting in aggregate formation. Whether other proteolytic pathways are involved in the pathogenesis of MFM deserves future investigations.