Integrated proteomic and transcriptomic profiling identifies aberrant gene and protein expression in the sarcomere, mitochondrial complex I, and the extracellular matrix in Warmblood horses with myofibrillar myopathy

Background Myofibrillar myopathy in humans causes protein aggregation, degeneration, and weakness of skeletal muscle. In horses, myofibrillar myopathy is a late-onset disease of unknown origin characterized by poor performance, atrophy, myofibrillar disarray, and desmin aggregation in skeletal muscle. This study evaluated molecular and ultrastructural signatures of myofibrillar myopathy in Warmblood horses through gluteal muscle tandem-mass-tag quantitative proteomics (5 affected, 4 control), mRNA-sequencing (8 affected, 8 control), amalgamated gene ontology analyses, and immunofluorescent and electron microscopy. Results We identified 93/1533 proteins and 47/27,690 genes that were significantly differentially expressed. The top significantly differentially expressed protein CSRP3 and three other differentially expressed proteins, including, PDLIM3, SYNPO2, and SYNPOL2, are integrally involved in Z-disc signaling, gene transcription and subsequently sarcomere integrity. Through immunofluorescent staining, both desmin aggregates and CSRP3 were localized to type 2A fibers. The highest differentially expressed gene CHAC1, whose protein product degrades glutathione, is associated with oxidative stress and apoptosis. Amalgamated transcriptomic and proteomic gene ontology analyses identified 3 enriched cellular locations; the sarcomere (Z-disc & I-band), mitochondrial complex I and the extracellular matrix which corresponded to ultrastructural Z-disc disruption and mitochondrial cristae alterations found with electron microscopy. Conclusions A combined proteomic and transcriptomic analysis highlighted three enriched cellular locations that correspond with MFM ultrastructural pathology in Warmblood horses. Aberrant Z-disc mechano-signaling, impaired Z-disc stability, decreased mitochondrial complex I expression, and a pro-oxidative cellular environment are hypothesized to contribute to the development of myofibrillar myopathy in Warmblood horses. These molecular signatures may provide further insight into diagnostic biomarkers, treatments, and the underlying pathophysiology of MFM.

FLNC-Associated Myofibrillar Myopathy

ObjectiveTo determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM).MethodsWe performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis.ResultsEight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc.ConclusionsThis new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

Pathogenic Mutations and Putative Phenotype-Affecting Variants in Polish Myofibrillar Myopathy Patients

Myofibrillar myopathies (MFM) are heterogeneous hereditary muscle diseases with characteristic myopathological features of Z-disk dissolution and aggregates of its degradation products. The onset and progression of the disease are variable, with an elusive genetic background, and around half of the cases lacking molecular diagnosis. Here, we attempted to establish possible genetic foundations of MFM by performing whole exome sequencing (WES) in eleven unrelated families of 13 patients clinically diagnosed as MFM spectrum. A filtering strategy aimed at identification of variants related to the disease was used and included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of muscle-expressed genes, and analysis of the disease-associated interactome. Genetic diagnosis was possible in eight out of eleven cases. Putative causative mutations were found in the DES (two cases), CRYAB, TPM3, and SELENON (four cases) genes, the latter typically presenting with a rigid spine syndrome. Moreover, a variety of additional, possibly phenotype-affecting variants were found. These findings indicate a markedly heterogeneous genetic background of MFM and show the usefulness of next generation sequencing in the identification of disease-associated mutations. Finally, we discuss the emerging concept of variant load as the basis of phenotypic heterogeneity.

A novel recessive mutation affecting DNAJB6a causes myofibrillar myopathy

Mutations in the DNAJB6 gene have been identified as rare causes of myofibrillar myopathies. However, the underlying pathophysiologica mechanisms remain elusive. DNAJB6 has two known isoforms, including the nuclear isoform DNAJB6a and the cytoplasmic isoform DNAJB6b, which was thought to be the pathogenic isoform. Here, we report a novel recessive mutation c.695_699del (p. Val 232 Gly fs*7) in the DNAJB6 gene, associated with an apparently recessively inherited late onset distal myofibrillar myopathy in a Chinese family. Notably, the novel mutation localizes to exon 9 and uniquely encodes DNAJB6a. We further identified that this mutation decreases the mRNA and protein levels of DNAJB6a and results in an age-dependent recessive toxic effect on skeletal muscle in knock-in mice. Moreover, the mutant DNAJB6a showed a dose-dependent anti-aggregation effect on polyglutamine-containing proteins in vitro. Taking together, these findings reveal the pathogenic role of DNAJB6a insufficiency in myofibrillar myopathies and expand upon the molecular spectrum of DNAJB6 mutations.

Cardiac Filaminopathies: Illuminating the Divergent Role of Filamin C Mutations in Human Cardiomyopathy

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.