Distinctive chaperonopathy in skeletal muscle associated with the dominant variant in DNAJB4.

DnaJ homolog, subfamily B, member 4, a member of the heat shock protein 40 chaperones encoded by DNAJB4, is highly expressed in myofibers. We identified a heterozygous c.270 T > A (p.F90L) variant in DNAJB4 in a family with a dominantly inherited distal myopathy, in which affected members have specific features on muscle pathology represented by the presence of cytoplasmic inclusions and the accumulation of desmin, p62, HSP70, and DNAJB4 predominantly in type 1 fibers. Both Dnajb4F90L knockin and knockout mice developed muscle weakness and recapitulated the patient muscle pathology in the soleus muscle, where DNAJB4 has the highest expression. These data indicate that the identified variant is causative, resulting in defective chaperone function and selective muscle degeneration in specific muscle fibers. This study demonstrates the importance of DNAJB4 in skeletal muscle proteostasis by identifying the associated chaperonopathy.

Desmin forms toxic, seeding-competent amyloid aggregates that persist in muscle fibers.

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance.

UNLABELLED: Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. SIGNIFICANCE STATEMENT: Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.

Myofibrillar disruption and RNA-binding protein aggregation in a mouse model of limb-girdle muscular dystrophy 1D.

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.

mTOR dysfunction contributes to vacuolar pathology and weakness in valosin-containing protein associated inclusion body myopathy.

Autophagy is dysfunctional in many degenerative diseases including myopathies. Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM) associated with Paget's disease of the bone, fronto-temporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS). VCP is necessary for protein degradation via the proteasome and lysosome. IBMPFD/ALS mutations in VCP disrupt autophagosome and endosome maturation resulting in vacuolation, weakness and muscle atrophy. To understand the regulation of autophagy in VCP-IBM muscle, we examined the AKT/FOXO3 and mammalian target of rapamycin (mTOR) pathways. Basal Akt and FOXO3 phosphorylation was normal. In contrast, the phosphorylation of mTOR targets was decreased. Consistent with this, global protein translation was diminished and autophagosome biogenesis was increased in VCP-IBM muscle. Further mTORC1 inhibition with rapamycin hastened weakness, atrophy and vacuolation in VCP-IBM mice. This was accompanied by the accumulation of autophagic substrates such as p62, LC3II and ubiquitinated proteins. The decrease in mTOR signaling was partially rescued by insulin and to a lesser extent by amino acid (AA) stimulation in VCP-IBM muscle. Cells expressing catalytically inactive VCP or treated with a VCP inhibitor also failed to activate mTOR upon nutrient stimulation. Expression of a constitutively active Rheb enhanced mTOR activity and increased the fiber size in VCP-IBM mouse skeletal muscle. These studies suggest that VCP mutations may disrupt mTOR signaling and contribute to IBMPFD/ALS disease pathogenesis. Treatment of some autophagic disorders with mTOR inhibitors such as rapamycin may worsen disease.

DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1.

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in vitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as in vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional in vivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism.

Skeletal muscle is the major site of glucose utilization in mammals integrating serum glucose clearance with mitochondrial respiration. To mechanistically elucidate the roles of iPLA2γ in skeletal muscle mitochondria, we generated a skeletal muscle-specific calcium-independent phospholipase A2γ knockout (SKMiPLA2γKO) mouse. Genetic ablation of skeletal muscle iPLA2γ resulted in pronounced muscle weakness, muscle atrophy, and increased blood lactate resulting from defects in mitochondrial function impairing metabolic processing of pyruvate and resultant bioenergetic inefficiency. Mitochondria from SKMiPLA2γKO mice were dysmorphic displaying marked changes in size, shape, and interfibrillar juxtaposition. Mitochondrial respirometry demonstrated a marked impairment in respiratory efficiency with decreases in the mass and function of oxidative phosphorylation complexes and cytochrome c. Further, a pronounced decrease in mitochondrial membrane potential and remodeling of cardiolipin molecular species were prominent. Collectively, these alterations prevented body weight gain during high-fat feeding through enhanced glucose disposal without efficient capture of chemical energy thereby altering whole-body bioenergetics.

Neuronal VCP loss of function recapitulates FTLD-TDP pathology.

The pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle.

Differentiated tissue is particularly vulnerable to alterations in protein and organelle homeostasis. The essential protein VCP, mutated in hereditary inclusion body myopathy, amyotrophic lateral sclerosis and frontotemporal dementia, is critical for efficient clearance of misfolded proteins and damaged organelles in dividing cells, but its role in terminally differentiated tissue affected by disease mutations is less clear. To understand the relevance of VCP in differentiated tissue, we inactivated it in skeletal muscle of adult mice. Surprisingly, knockout muscle demonstrated a necrotic myopathy with increased macroautophagic/autophagic proteins and damaged lysosomes. This was not solely due to a defect in autophagic degradation because age-matched mice with muscle inactivation of the autophagy essential protein, ATG5, did not demonstrate a myopathy. Notably, myofiber necrosis was preceded by upregulation of LGALS3/Galectin-3, a marker of damaged lysosomes, and TFEB activation, suggesting early defects in the lysosomal system. Consistent with that, myofiber necrosis was recapitulated by chemical induction of lysosomal membrane permeabilization (LMP) in skeletal muscle. Moreover, TFEB was activated after LMP in cells, but activation and nuclear localization of TFEB persisted upon VCP inactivation or disease mutant expression. Our data identifies VCP as central mediator of both lysosomal clearance and biogenesis in skeletal muscle. Abbreviations: AAA: ATPases Associated with diverse cellular Activities; TUBA1A/α-tubulin: tubulin alpha 1a; ATG5: autophagy related 5; ATG7: autophagy related 7; ACTA1: actin alpha 1, skeletal muscle; CLEAR: coordinated lysosomal expression and regulation; CTSB/D: cathepsin B/D; Ctrl: control; DAPI: diamidino-2-phenylindole; EBSS: Earle's balanced salt solution; ELDR: endolysosomal damage response; ESCRT: endosomal sorting complexes required for transport; Gastroc/G: gastrocnemius; H&E: hematoxylin and eosin; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IBMPFD/ALS: inclusion body myopathy associated with Paget disease of the bone, frontotemporal dementia and amyotrophic lateral sclerosis; i.p.: intraperitoneal; LAMP1/2: lysosomal-associated membrane protein 1/2; LLOMe: Leu-Leu methyl ester hydrobromide; LGALS3/Gal3: galectin 3; LMP: lysosomal membrane permeabilization; MTOR: mechanistic target of rapamycin kinase; MYL1: myosin light chain 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSP: multisystem proteinopathy; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; Quad/Q: quadriceps; RHEB: Ras homolog, mTORC1 binding; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TA: tibialis anterior; siRNA: small interfering RNA; SQSTM1/p62, sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBS: Tris-buffered saline; TXFN, tamoxifen; UBXN6/UBXD1: UBX domain protein 6; VCP: valosin containing protein; WT: wild-type.

Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D.

OBJECTIVE: To understand DNAJB6's function in skeletal muscle and identify therapeutic targets for limb-girdle muscular dystrophy 1D (LGMD1D). METHODS: DNAJB6 knockout (KO) myoblasts were generated with Crispr/cas9 technology, and differentially accumulated proteins were identified using stable isotope labeling, followed by quantitative mass spectrometry. Cultured KO myotubes and mouse muscle from DNAJB6b-WT or DNAJB6b-F93L mice were analyzed using histochemistry, immunohistochemistry, and immunoblot. Mouse functional strength measures included forelimb grip strength and inverted wire hang. RESULTS: DNAJB6 inactivation leads to the accumulation of sarcomeric proteins and hypertrophic myotubes with an enhanced fusion index. The increased fusion in DNAJB6 KO myotubes correlates with diminished glycogen synthase kinase-ß (GSK3ß) activity. In contrast, LGMD1D mutations in DNAJB6 enhance GSK3ß activation and suppress ß-catenin and NFAT3c signaling. GSK3ß inhibition with lithium chloride improves muscle size and strength in an LGMD1D preclinical mouse model. CONCLUSIONS: Our results suggest that DNAJB6 facilitates protein quality control and negatively regulates myogenic signaling. In addition, LGMD1D-associated DNAJB6 mutations inhibit myogenic signaling through augmented GSK3ß activity. GSK3ß inhibition with lithium chloride may be a therapeutic option in LGMD1D.

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination.

p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62's UBA domain at lysine 420 may regulate p62's function and be disrupted in p62-associated disease.

Autophagic vacuolar pathology in desminopathies.

Autophagic vacuolar myopathies are an emerging group of muscle diseases with common pathologic features. These include autophagic vacuoles containing both lysosomal and autophagosomal proteins sometimes lined with sarcolemmal proteins such as dystrophin. These features have been most clearly described in patients with Danon's disease due to LAMP2 deficiency and X-linked myopathy with excessive autophagy (XMEA) due to mutations in VMA21. Disruptions of these proteins lead to lysosomal dysfunction and subsequent autophagic vacuolar pathology. We performed whole exome sequencing on two families with autosomal dominantly inherited myopathies with autophagic vacuolar pathology and surprisingly identified a p.R454W tail domain mutation and a novel p.S6W head domain mutation in desmin, DES. In addition, re-evaluation of muscle tissue from another family with a novel p.I402N missense DES mutation also identified autophagic vacuoles. We suggest that autophagic vacuoles may be an underappreciated pathology present in desminopathy patient muscle. Moreover, autophagic vacuolar pathology can be due to genetic etiologies unrelated to primary defects in the lysosomes or autophagic machinery. Specifically, cytoskeletal derangement and the accumulation of aggregated proteins such as desmin may activate the autophagic system leading to the pathologic features of an autophagic vacuolar myopathy.

Targeted sequencing and identification of genetic variants in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) has clinical, pathologic and pathomechanistic overlap with some inherited muscle and neurodegenerative disorders. In this study, DNA from 79 patients with sIBM was collected and the sequencing of 38 genes associated with hereditary inclusion body myopathy (IBM), myofibrillar myopathy, Emery-Dreifuss muscular dystrophy, distal myopathy, amyotrophic lateral sclerosis and dementia along with C9orf72 hexanucleotide repeat analysis was performed. No C9orf72 repeat expansions were identified, but; 27 rare (minor allele frequency <1%) missense coding variants in several other genes were identified. One patient carried a p.R95C missense mutation in VCP and another carried a previously reported p.I27V missense mutation in VCP. Mutations in VCP cause IBM associated with Paget's disease of the bone (PDB) and fronto-temporal dementia (IBMPFD). Neither patient had a family history of weakness or manifested other symptoms reported with VCP mutations such as PDB or dementia. In vitro analysis of these VCP variants found that they both disrupted autophagy similar to other pathogenic mutations. Although no clear genetic etiology has been implicated in sIBM pathogenesis, our study suggests that genetic evaluation in sIBM may be clinically meaningful and lend insight into its pathomechanism.

SQSTM1 splice site mutation in distal myopathy with rimmed vacuoles.

OBJECTIVE: To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy. METHODS: We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies. RESULTS: Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization. CONCLUSIONS: SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes.

Increased autophagy accelerates colchicine-induced muscle toxicity.

Colchicine treatment is associated with an autophagic vacuolar myopathy in human patients. The presumed mechanism of colchicine-induced myotoxicity is the destabilization of the microtubule system that leads to impaired autophagosome-lysosome fusion and the accumulation of autophagic vacuoles. Using the MTOR inhibitor rapamycin we augmented colchicine's myotoxic effect by increasing the autophagic flux; this resulted in an acute myopathy with muscle necrosis. In contrast to myonecrosis induced by cardiotoxin, myonecrosis induced by a combination of rapamycin and colchicine was associated with accumulation of autophagic substrates such as LC3-II and SQSTM1; as a result, autophagic vacuoles accumulated in the center of myofibers, where LC3-positive autophagosomes failed to colocalize with the lysosomal protein marker LAMP2. A similar pattern of central LC3 accumulation and myonecrosis is seen in human patients with colchicine myopathy, many of whom have been treated with statins (HMGCR/HMG-CoA reductase inhibitors) in addition to colchicine. In mice, cotreatment with colchicine and simvastatin also led to muscle necrosis and LC3 accumulation, suggesting that, like rapamycin, simvastatin activates autophagy. Consistent with this, treatment of mice with four different statin medications enhanced autophagic flux in skeletal muscle in vivo. Polypharmacy is a known risk factor for toxic myopathies; our data suggest that some medication combinations may simultaneously activate upstream autophagy signaling pathways while inhibiting the degradation of these newly synthesized autophagosomes, resulting in myotoxicity.