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Background: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. Methods: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. Results: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. Conclusions: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Background Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p. R350P. Methods and results Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 +/- 2.9%, HET-sham: 64.5 +/- 4.7%, WT-TAC: 63.5 +/- 4.9%, HET-TAC: 55.7 +/- 5.4%; p < 0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 +/- 2385 mu l/min, HET sham: 10391 +/- 1349 mu l/min, WT-TAC: 8097 +/- 1903 mu l/min, HET-TAC: 5793 +/- 2517 mu l/min; p< 0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 +/- 406, HET-sham: 13526 +/- 781, WT-TAC: 11155 +/- 3315, HET-TAC: 8649 +/- 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. Conclusion Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

Aims We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. Methods We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. Results We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of alpha B-crystallin and Hsp27. Both alpha B-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. Conclusions Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.

Mutations of the human valosin-containing protein, p97 (VCP) and Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex genes cause motor neuron and cognitive impairment disorders. Here, we analyzed a cohort of German patients with sporadic amyotrophic lateral sclerosis and frontotemporal lobar degeneration comorbidity (ALS/FTD) for VCP and WASH complex gene mutations. Next-generation panel sequencing of VCP, WASH1, FAM21C, CCDC53, SWIP, strumpellin, F-actin capping protein of muscle Z-line alfa 1 (CAPZA1), and CAPZB genes was performed in 43 sporadic ALS/FTD patients. Subsequent analyses included Sanger sequencing, in silico analyses, real-time PCR, and CCDC53 immunoblotting. We identified 1 patient with the heterozygous variant c.26C>T in CAPZA1, predicted to result in p.Ser9Leu, and a second with the heterozygous start codon variant c.2T>C in CCDC53. In silico analysis predicted structural changes in the N-terminus of CAPZα1, which may interfere with CAPZα:CAPZβ dimerization. Though the translation initiation codon of CCDC53 is mutated, real-time PCR and immunoblotting did neither reveal any evidence for a CCDC53 haploinsufficiency nor for aberrant CCDC53 protein species. Moreover, a disease-causing C9orf72 repeat expansion mutation was later on identified in this patient. Thus, with the exception of a putatively pathogenic heterozygous c.26C>T CAPZA1 variant, our genetic analysis did not reveal mutations in VCP and the remaining WASH complex subunits.

Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain (“head”) of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments.