The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues.

AIMS: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. METHODS: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. RESULTS: Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. CONCLUSIONS: Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

N471D WASH complex subunit strumpellin knock-in mice display mild motor and cardiac abnormalities and BPTF and KLHL11 dysregulation in brain tissue.

AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice.

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.

Making sense of missense variants in TTN-related congenital myopathies.

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Growing Old Too Early: Skeletal Muscle Single Fiber Biomechanics in Ageing R349P Desmin Knock-in Mice Using the MyoRobot Technology.

Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.

23 Na MRI depicts early changes in ion homeostasis in skeletal muscle tissue of patients with duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a hereditary neuromuscular disease leading to progressive muscle wasting. Since there is a need for MRI variables that serve as early sensitive indicators of response to treatment, several quantitative MRI methods have been suggested for disease monitoring. PURPOSE: To evaluate the potential of sodium (23 Na) and proton (1 H) MRI methods to assess early pathological changes in skeletal muscle of DMD. STUDY TYPE: Prospective clinical study. POPULATION: 23 Na and 1 H MRI of the right leg were performed in 13 patients with DMD (age 7.8 ± 2.4) and 14 healthy boys (age 9.5 ± 2.2). FIELD STRENGTH/SEQUENCE: 3 T including a multiecho-spin-echo sequence, diffusion-weighted sequences, 1 H spectroscopy, 3-pt Dixon, and 23 Na ultrashort echo time sequences. ASSESSMENT: We obtained water T2 maps, fat fraction (FF), pH, and diffusion properties of the skeletal muscle tissue. Moreover, total tissue sodium concentration (TSC) was calculated from the 23 Na sequence. Intracellular-weighted 23 Na signal (ICwS) was derived from 23 Na inversion-recovery imaging. STATISTICAL TESTS: Results from DMD patients and controls were compared using Wilcoxon rank-sum tests and repeated analysis of variance (ANOVA). Spearman-rank correlations and area under the curve (AUC) were calculated to assess the performance of the different MRI methods to distinguish dystrophic from healthy muscle tissue. RESULTS: FF, water T2 , and pH were higher in DMD patients (0.07 ± 0.03, 39.4 ± 0.8 msec, 7.06 ± 0.03, all P < 0.05) than in controls (0.02 ± 0.01, 36.0 ± 0.4 msec, 7.03 ± 0.02). No difference was observed in diffusion properties. TSC (26.0 ± 1.3 mM, P < 0.05) and ICwS (0.69 ± 0.05 a.u., P < 0.05) were elevated in DMD (controls: 16.5 ± 1.3 mM and 0.47 ± 0.04 a.u.). The ICwS was frequently abnormal in DMD even when water T2 , FF, and pH were in the normal range. 23 Na MRI showed higher AUC values in comparison to the 1 H methods. DATA CONCLUSION: Sodium anomalies were regularly observed in patients with DMD compared with controls, and were present even in absence of fatty degenerative changes and water T2 increases. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:1103-1113.

Camptocormia as the presenting symptom in sporadic late onset nemaline myopathy: a case report.

BACKGROUND: Camptocormia has been reported in a plethora of diseases comprising disorders of the central nervous system, the peripheral nervous system, and the neuromuscular junction as well as hereditary and acquired myopathies. In sporadic late onset nemaline myopathy concomitant axial myopathy is common, but reports about camptocormia as the only presenting symptom in this condition are very rare. Notably, sporadic late onset nemaline myopathy is a potentially treatable condition in particular when associated with monoclonal gammopathy of unknown significance, HIV or rheumatological disorders. CASE PRESENTATION: We report the case of a 62-year-old female patient, who presented with slowly progressive camptocormia. Comprehensive work-up including neurological work-up, laboratory tests, MR-imaging, muscle biopsy and genetic testing led to the diagnosis of sporadic late onset nemaline myopathy. CONCLUSIONS: Our case report highlights that sporadic late onset nemaline myopathy has to be considered in patients presenting with isolated camptocormia and comprehensive work-up of camptocormia is mandatory to ascertain the individual diagnosis, especially in consideration of treatable conditions.

The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?

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.

[Inclusion Body Myopathy, Paget's Disease, and Fronto-temporal Dementia: a VCP-related Multi-systemic Proteinopathy]. / Einschlusskörpermyopathie, Paget-Krankheit und frontotemporale Demenz: eine VCP-bedingte, multisystemische Proteinopathie.

Mutations of the human VCP gene, which encodes the V: alosin C: ontaining P: rotein (synonyms: p97, TER ATPase), are associated with various multi-systemic protein aggregation diseases. We report on a patient with progressive myopathy and incipient cognitive deficits. A diagnostic muscle biopsy revealed an inclusion body myopathy with protein aggregates. Magnetic resonance imaging and F18-positron-emission-tomography disclosed a fronto-temporal atrophy and glucose hypometabolism of the frontal and temporal lobes, respectively. Based on the clinical findings, a genetic analysis was performed which revealed a heterozygous c.277C>T (p.Arg93Cys) mutation of the VCP gene, thus confirming the diagnosis of IBMPFD (I: nclusion B: ody M: yopathie with P: aget Disease of the Bones and F: ronto-temporal D: ementia).

Myofibrillar instability exacerbated by acute exercise in filaminopathy.

Filamin C (FLNC) mutations in humans cause myofibrillar myopathy (MFM) and cardiomyopathy, characterized by protein aggregation and myofibrillar degeneration. We generated the first patient-mimicking knock-in mouse harbouring the most common disease-causing filamin C mutation (p.W2710X). These heterozygous mice developed muscle weakness and myofibrillar instability, with formation of filamin C- and Xin-positive lesions streaming between Z-discs. These lesions, which are distinct from the classical MFM protein aggregates by their morphology and filamentous appearance, were greatly increased in number upon acute physical exercise in the mice. This pathology suggests that mutant filamin influences the mechanical stability of myofibrillar Z-discs, explaining the muscle weakness in mice and humans. Re-evaluation of biopsies from MFM-filaminopathy patients with different FLNC mutations revealed a similar, previously unreported lesion pathology, in addition to the classical protein aggregates, and suggested that structures previously interpreted as aggregates may be in part sarcomeric lesions. We postulate that these lesions define preclinical disease stages, preceding the formation of protein aggregates.

Stroke in Duchenne Muscular Dystrophy: A Retrospective Longitudinal Study in 54 Patients.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD) is the most frequent skeletal muscle myopathy. Nearly all patients develop cardiomyopathy in their second decade of life. The purpose of this study was to evaluate the frequency, cause, and outcome of stroke in a German cohort of patients with DMD. METHODS: Retrospective analysis of medical records of 54 DMD patients, who lived in a regional facility for handicapped people (Wichernhaus Altdorf, Germany) between 1963 and 2013. RESULTS: Fifty-four DMD patients were followed up for 7.4 years on average. Mean age at admission and discharge from the long-term care facility or death were 11.4 and 18.8 years, respectively. Covering a total observation period of 400 patient-years, we identified 4 DMD patients with juvenile arterial ischemic strokes. Off-label systemic thrombolysis in 2 patients resulted in a nearly complete regression of stroke-related symptoms, but 1 patient died of septic pneumonia and cardiac failure 24 days after thrombolysis therapy. In the other 2 patients, who had their ischemic strokes in 1994 and 1998, severe infarction-related symptoms persisted, and 1 patient died 13 days later. DMD-associated cardiomyopathy without evidence of atrial fibrillation was the only risk factor for ischemic stroke in all patients. CONCLUSIONS: This study indicates an increased risk for ischemic strokes in DMD patients. Regular cardiological assessment of all DMD patients is mandatory to evaluate the individual risk profile for cardioembolic events and to adapt therapeutic strategies.

New aspects of myofibrillar myopathies.

PURPOSE OF REVIEW: Myofibrillar myopathies (MFMs) are hereditary muscle disorders characterized by distinct histopathological features. This review provides an overview of recent research with respect to new disease genes, clinical phenotypes, insights into pathomechanisms and therapeutic strategies. RECENT FINDINGS: Beyond the known disease genes DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1 and TTN, mutations in PLEC, ACTA1, HSPB8 and DNAJB6 have also been associated with a MFM phenotype. Proteomic analysis revealed new information about the composition of protein aggregates in myotilinopathy and identified a new diagnostic marker. New animal models mirror central aspects of MFM pathology and novel therapeutic strategies for treatment of MFM were evaluated in cell and animal models. SUMMARY: MFMs are an expanding and numerically significant group of protein aggregate diseases with marked clinical and genetic heterogeneity. Though no specific therapy is currently available, the generation of patient-mimicking cell and animal models now paves the way for the preclinical evaluation of novel therapeutic strategies.

Mutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue.

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.

In vivo characterization of human myofibrillar myopathy genes in zebrafish.

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.

VCP and PSMF1: Antagonistic regulators of proteasome activity.

Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.

The toxic effect of R350P mutant desmin in striated muscle of man and mouse.

Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy.

Hereditary myopathy with early respiratory failure: occurrence in various populations.

OBJECTIVE: Several families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying cause in these families, and to characterise the phenotype in our large cohort. METHODS: DNA samples of all currently known families with HMERF without molecular genetic cause were obtained from 12 families in seven different countries. Clinical, histopathological and muscle imaging data were collected and five biopsy samples made available for further immunohistochemical studies. Genotyping, exome sequencing and Sanger sequencing were used to identify and confirm sequence variations. RESULTS: All patients with clinical diagnosis of HMERF were genetically solved by five different titin mutations identified. One mutation has been reported while four are novel, all located exclusively in the FN3 119 domain (A150) of A-band titin. One of the new mutations showed semirecessive inheritance pattern with subclinical myopathy in the heterozygous parents. Typical clinical features were respiratory failure at mid-adulthood in an ambulant patient with very variable degree of muscle weakness. Cytoplasmic bodies were retrospectively observed in all muscle biopsy samples and these were reactive for myofibrillar proteins but not for titin. CONCLUSIONS: We report an extensive collection of families with HMERF with five different mutations in exon 343 of TTN, which establishes this exon as the primary target for molecular diagnosis of HMERF. Our relatively large number of new families and mutations directly implies that HMERF is not extremely rare, not restricted to Northern Europe and should be considered in undetermined myogenic respiratory failure.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria.

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.