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PLoS Genetics Nov 2020Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart...
Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart failure, as well as myofibrillar myopathy (MFM), which is characterized by protein aggregation and myofibrillar disintegration in skeletal muscle cells. Here, we generated a CRISPR/Cas9-induced Bag3 knockout zebrafish line and found the complete preservation of heart and skeletal muscle structure and function during embryonic development, in contrast to morpholino-mediated knockdown of Bag3. Intriguingly, genetic compensation, a process of transcriptional adaptation which acts independent of protein feedback loops, was found to prevent heart and skeletal muscle damage in our Bag3 knockout model. Proteomic profiling and quantitative real-time PCR analyses identified Bag2, another member of the Bag protein family, significantly upregulated on a transcript and protein level in bag3-/- mutants. This implied that the decay of bag3 mutant mRNA in homozygous bag3-/- embryos caused the transcriptional upregulation of bag2 expression. We further demonstrated that morpholino-mediated knockdown of Bag2 in bag3-/- embryos evoked severe functional and structural heart and skeletal muscle defects, which are similar to Bag3 morphants. However, Bag2 knockdown in bag3+/+ or bag3+/- embryos did not result in (cardio-)myopathy. Finally, we found that inhibition of the nonsense-mediated mRNA decay (NMD) machinery by knockdown of upf1, an essential NMD factor, caused severe heart and skeletal muscle defects in bag3-/- mutants due to the blockade of transcriptional adaptation of bag2 expression. Our findings provide evidence that genetic compensation might vitally influence the penetrance of disease-causing bag3 mutations in vivo.
Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cardiomyopathies; Cardiomyopathy, Dilated; Disease Models, Animal; Heart Failure; Molecular Chaperones; Muscle Fibers, Skeletal; Muscular Diseases; Mutation; Myocardium; Myopathies, Structural, Congenital; Phenotype; Proteomics; Zebrafish; Zebrafish Proteins
PubMed: 33137814
DOI: 10.1371/journal.pgen.1009088 -
MedRxiv : the Preprint Server For... Feb 2024Myofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of...
Myofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of MFM6 is p.Pro209Leu mutation (rs121918312-T) in the gene, which generates the disease outcomes in a dominant fashion. Since the consequences of the mutation are strong and rapidly progressing, most MFM6 patients are due to mutation. There are no effective treatments for MFM6 despite its well-known genetic cause. Given p.Pro209Leu mutation is dominant, regenerative medicine approaches employing orthologous stem cells in which mutant is inactivated offer a promising avenue. Here, we developed personalized allele-specific CRISPR-Cas9 strategies capitalizing on PAM-altering SNP and PAM-proximal SNP. In order to identify the disease chromosome carrying the mutation in our two affected individuals, haplotype phasing through cloning-sequencing was performed. Based on the sequence differences between mutant and normal , we developed personalized allele-specific CRISPR-Cas9 strategies to selectively inactivate the mutant allele 1) by preventing the transcription of the mutant and 2) by inducing nonsense-mediated decay (NMD) of mutant mRNA. Subsequent experimental validation in patient-derived induced pluripotent stem cell (iPSC) lines showed complete allele specificities of our CRISPR-Cas9 strategies and molecular consequences attributable to inactivated mutant . In addition, mutant allele-specific CRISPR-Cas9 targeting did not alter the characteristics of iPSC or the capacity to differentiate into cardiomyocytes. Together, our data demonstrate the feasibility and potential of personalized allele-specific CRISPR-Cas9 approaches to selectively inactivate the mutant to generate cell resources for regenerative medicine approaches for MFM6.
PubMed: 38352343
DOI: 10.1101/2024.02.03.24302252 -
Acta Myologica : Myopathies and... Mar 2019Tripartite motif-containing protein 32 () is a member of the TRIM ubiquitin E3 ligases which ubiquitinates different substrates in muscle including sarcomeric proteins....
Tripartite motif-containing protein 32 () is a member of the TRIM ubiquitin E3 ligases which ubiquitinates different substrates in muscle including sarcomeric proteins. Mutations in are associated with Limb-Girdle Muscular Dystrophy 2H. In a 66 old woman with disto-proximal myopathy, we identified a novel homozygous mutation of gene c.1781G > A (p. Ser594Asn) localised in the c-terminus NHL domain. Mutations of this domain have been also associated to Sarcotubular Myopathy (STM), a form of distal myopathy with peculiar features in muscle biopsy, now considered in the spectrum of LGMD2H. Muscle biopsy revealed severe abnormalities of the myofibrillar network with core like areas, lobulated fibres, whorled fibres and multiple vacuoles. Desmin and Myotilin stainings also pointed to accumulation as in Myofibrillar Myopathy. This report further confirms that STM and LGMD2H represent the same disorder and suggests to consider mutations in the genetic diagnosis of Sarcotubular Myopathy and Myofibrillar Myopathy.
Topics: Aged; Female; Humans; Muscular Dystrophies, Limb-Girdle; Mutation; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 31309175
DOI: No ID Found -
Biochemical and Biophysical Research... May 2015Myofibrillar myopathies (MFM) are progressive diseases of human heart and skeletal muscle with a severe impact on life quality and expectancy of affected patients....
Myofibrillar myopathies (MFM) are progressive diseases of human heart and skeletal muscle with a severe impact on life quality and expectancy of affected patients. Although recently several disease genes for myofibrillar myopathies could be identified, today most genetic causes and particularly the associated mechanisms and signaling events that lead from the mutation to the disease phenotype are still mostly unknown. To assess whether the zebrafish is a suitable model system to validate MFM candidate genes using targeted antisense-mediated knock-down strategies, we here specifically inactivated known human MFM disease genes and evaluated the resulting muscular and cardiac phenotypes functionally and structurally. Consistently, targeted ablation of MFM genes in zebrafish led to compromised skeletal muscle function mostly due to myofibrillar degeneration as well as severe heart failure. Similar to what was shown in MFM patients, MFM gene-deficient zebrafish showed pronounced gene-specific phenotypic and structural differences. In summary, our results indicate that the zebrafish is a suitable model to functionally and structurally evaluate novel MFM disease genes in vivo.
Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Gene Knockdown Techniques; Genetic Predisposition to Disease; Heart Failure; Humans; Muscle, Skeletal; Myocardium; Myopathies, Structural, Congenital; Zebrafish
PubMed: 25866181
DOI: 10.1016/j.bbrc.2015.03.149 -
Journal of Clinical Medicine Feb 2021Over the past decades, there has been tremendous progress in understanding genetic alterations that can result in different phenotypes of human cardiomyopathies. More... (Review)
Review
Over the past decades, there has been tremendous progress in understanding genetic alterations that can result in different phenotypes of human cardiomyopathies. More than a thousand mutations in various genes have been identified, indicating that distinct genetic alterations, or combinations of genetic alterations, can cause either hypertrophic (HCM), dilated (DCM), restrictive (RCM), or arrhythmogenic cardiomyopathies (ARVC). Translation of these results from "bench to bedside" can potentially group affected patients according to their molecular etiology and identify subclinical individuals at high risk for developing cardiomyopathy or patients with overt phenotypes at high risk for cardiac deterioration or sudden cardiac death. These advances provide not only mechanistic insights into the earliest manifestations of cardiomyopathy, but such efforts also hold the promise that mutation-specific pathophysiology might result in novel "personalized" therapeutic possibilities. Recently, the FLNC gene encoding the sarcomeric protein filamin C has gained special interest since FLNC mutations were found in several distinct and possibly overlapping cardiomyopathy phenotypes. Specifically, mutations in FLNC were initially only linked to myofibrillar myopathy (MFM), but are now increasingly found in various forms of human cardiomyopathy. FLNC thereby represents another example for the complex genetic and phenotypic continuum of these diseases.
PubMed: 33557094
DOI: 10.3390/jcm10040577 -
Anatomical Record (Hoboken, N.J. : 2007) Sep 2014Several missense mutations in the Z-band protein, myotilin, have been implicated in human muscle diseases such as myofibrillar myopathy, spheroid body myopathy, and...
Several missense mutations in the Z-band protein, myotilin, have been implicated in human muscle diseases such as myofibrillar myopathy, spheroid body myopathy, and distal myopathy. Recently, we have reported the cloning of chicken myotilin cDNA. In this study, we have investigated the expression of myotilin in cross-striated muscles from developing chicken by qRT-PCR and in situ hybridizations. In situ hybridization of embryonic stages shows myotilin gene expression in heart, somites, neural tissue, eyes and otocysts. RT-PCR and qRT-PCR data, together with in situ hybridization results point to a biphasic transcriptional pattern for MYOT gene during early heart development with maximum expression level in the adult. In skeletal muscle, the expression level starts decreasing after embryonic day 20 and declines in the adult skeletal muscles. Western blot assays of myotilin in adult skeletal muscle reveal a decrease in myotilin protein compared with levels in embryonic skeletal muscle. Our results suggest that MYOT gene may undergo transcriptional activation and repression that varies between tissues in developing chicken. We believe this is the first report of the developmental regulation on myotilin expression in non-mammalian species.
Topics: Animals; Chick Embryo; Chickens; Connectin; Gene Expression Regulation, Developmental; Heart; Muscle, Skeletal; Myocytes, Cardiac; RNA, Messenger; Time Factors; Transcription, Genetic; Transcriptional Activation
PubMed: 25125173
DOI: 10.1002/ar.22964 -
Acta Neuropathologica Communications Feb 2016Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular...
INTRODUCTION
Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy. After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy.
RESULTS
Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, αB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31.
CONCLUSIONS
Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 - a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis.
Topics: Adult; Family Health; Female; Gene Expression Regulation; HSP40 Heat-Shock Proteins; Humans; Male; Middle Aged; Molecular Chaperones; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Mutation; Nerve Tissue Proteins; Young Adult
PubMed: 26847086
DOI: 10.1186/s40478-016-0276-9 -
The American Journal of Pathology Mar 2020BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy....
BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.
Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Cardiomyopathies; Disease Models, Animal; Genes, Dominant; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Muscle Fibers, Skeletal; Muscle, Skeletal; Mutation; Myopathies, Structural, Congenital; Phenotype
PubMed: 31953038
DOI: 10.1016/j.ajpath.2019.11.005 -
Neuromuscular Disorders : NMD Aug 2022The diagnosis of adult-onset genetic muscle diseases is challenging because of the diversity of clinical phenotypes, findings on muscle biopsy that may be nonspecific,...
The diagnosis of adult-onset genetic muscle diseases is challenging because of the diversity of clinical phenotypes, findings on muscle biopsy that may be nonspecific, and the large number of genetic causes. Even with thorough investigation, the diagnostic yield for genetic testing in these populations is very low, and the distinction from acquired conditions such as sporadic inclusion body myositis [sIBM] can also prove difficult. In this study, we analysed whole transcriptome data generated from RNA isolated from muscle biopsy tissues, from a cohort of 16 participants with sIBM and other histologic diagnoses. Our objective was to identify candidate RNA biomarkers that could be an adjunctive tool in differentiating these conditions. Principal component analysis was able to delineate the groups based on their histologic diagnoses. Gene ontology and pathway analyses demonstrated dysregulation of immune pathways in sIBM. In mitochondrial myopathy we observed upregulation of FGF21, GDF15, ASNS and TRIB3, which are known candidate biomarkers for mitochondrial myopathy. Novel findings included the identification of transcripts of unknown function that were dysregulated in myofibrillar myopathy [JPX], dystrophic changes [MEG3], and mitochondrial myopathy [GAS5]. We suggest future investigations with larger cohorts of participants to confirm the findings of this study, with further directed experiments to determine the role of novel transcripts in disease pathogenesis.
Topics: Biomarkers; Biopsy; Gene Expression Profiling; Humans; Muscle, Skeletal; Myositis, Inclusion Body; RNA
PubMed: 35850946
DOI: 10.1016/j.nmd.2022.04.009 -
Communications Biology Mar 2021Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins...
Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3 mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Disease Models, Animal; Down-Regulation; Filamins; HSC70 Heat-Shock Proteins; LIM Domain Proteins; Mechanotransduction, Cellular; Mice, Inbred C57BL; Mice, Transgenic; Muscle Contraction; Muscle Strength; Muscle, Skeletal; Myopathies, Structural, Congenital; Point Mutation; Protein Aggregates; Protein Aggregation, Pathological; Protein Kinase C-alpha; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Mice
PubMed: 33742095
DOI: 10.1038/s42003-021-01864-1