P.F.M. van der Ven

Structural basis for activation of the titin kinase domain during myofibrillogenesis

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titins only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine–aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P+ 1 loop, indicating that this tyrosine is a binding partner of the titinkinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle

Filamin C is the muscle isoform of a group of large actin-crosslinking proteins. On the one hand, filamin C is associated with the Z-disk of the myofibrillar apparatus and binds to myotilin; on the other hand, it interacts with the sarcoglycan complex at the sarcolemma. Filamin C may be involved in reorganizing the cytoskeleton in response to signalling events and in muscle it may, in addition, fulfill structural functions at the Z-disk. An examination of biopsies from patients with multi-minicore myopathy, central core myopathy and neurogenic target fibers with core-like target formations (TF) revealed strong reactivity of all the cores and target formations with two different anti-filamin C antibodies. In all three conditions, the immunoreactivity in the cores for filamin C was considerably stronger than that for desmin. Only for alphaB-crystallin were comparable levels of immunoreactivity detected. There was no difference in intensity for filamin C between the three pathological conditions. Thus, filamin C along with alphaB-crystallin is a strong and robust, but nonspecific marker of core formation. The reason why filamin C accumulates in cores is unclear at present, but we postulate that it may be critically involved in the chain of events eventually leading to myofibrillar degeneration.

Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy.

UNLABELLED Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p<0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder. BIOLOGICAL SIGNIFICANCE Our proteomic analysis provides essential new insights in the composition of pathological protein aggregates in skeletal muscle fibers of desminopathy patients. The results contribute to a better understanding of pathomechanisms in myofibrillar myopathies and provide the basis for hypothesis-driven studies. The detection of specific proteomic profiles in different myofibrillar myopathy subtypes indicates that proteomic analysis may become a useful tool in differential diagnosis of protein aggregate myopathies.

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

Objective: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. Methods: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. Results: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. Conclusions: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.

Xin-deficient mice display myopathy, impaired contractility, attenuated muscle repair and altered satellite cell functionality.

Xin is an F‐actin‐binding protein expressed during development of cardiac and skeletal muscle. We used Xin‐/‐ mice to determine the impact of Xin deficiency on different aspects of skeletal muscle health, including functionality and regeneration.

O.5 Mutations in MuRF1 and MuRF3 cause a novel protein aggregate myopathy and cardiomyopathy

MuRF1 and MuRF3 are microtubule-associated proteins that localize to the sarcomeric M-band and Z-line of striated muscle. They are E3 ubiquitin ligases that mediate degradation of sarcomeric proteins and participate in diverse biological functions including stabilisation of microtubules and myogenesis. Double knock-out mice for MuRF1 and MuRF3 developed cardiomyopathy and skeletal myopathy. Morphological features included myosin accumulation and aggregates of fragmented sarcomeres with preserved A-bands and M-lines. We studied a family with three affected members suffering from cardiomyopathy or cardioskeletal myopathy. We performed sequencing analysis of TRIM63 and TRIM54 encoding MuRF1 and MuRF3, respectively. Transfection of wild-type MuRF1 in cultured patient myoblast was performed to assess the rescue assay. To characterize the behaviour of TRIM54 mutation, control myoblasts were transfected with wild-type or mutant MuRF3. We identified a homozygous MuRF1 null mutation and heterozygous MuRF3 mutation in the patient with cardioskeletal myopathy. Myopathological features were highly reminiscent of that of MuRF1−/−/MuRF3−/− mice. Cultured myotubes from patient showed perturbed myofibrillogenesis and abnormal organization of the microtubule network. Transfection of wild-type MuRF1 in cultured patient myocytes rescued the phenotype. Transfection of control myoblasts with mutant MuRF3 induced the formation of irregular filamentous structures demonstrating the pathogenic effect of the MuRF3 mutation. We describe a novel protein aggregate myopathy and cardiomyopathy due to MuRF1 deficiency and a deleterious MuRF3 missense mutation. Unique morphological features including myosin accumulation, disrupted microtubule structures and fragmented sarcomeres with preserved A-bands and M-lines in the absence of thin filaments and Z-lines characterize the disease associated with combined MuRF1 and MuRF3 deficiency.

G.O.5 Deciphering protein aggregates in myofibrillar myopathies – A proteomics approach

Abstract Myofibrillar myopathies (MFM) encompass a genetic heterogenous group of muscle disorders characterized by formation of intracellular protein aggregates in skeletal muscle fibers. We applied a proteomic approach to decipher the aggregate composition in MFM subtypes with the aim to identify novel disease-relevant proteins, disease-specific proteomic profiles and new candidates for MFM-causing proteins. Muscle biopsies of 21 genetically clarified MFM patients (filaminopathy, myotilinopathy, desminopathy, ZASPopathy) were analyzed. Aggregates and intraindividual controls (muscle fibers without aggregates) were collected by laser microdissection. A label-free mass spectrometric approach was used for identification and relative quantification of proteins. A total of 588 different proteins were identified. Up to 193 proteins showed an accumulation in protein aggregates in the different MFM subtypes (ratio >1.8 compared to intraindividual controls). Many of these proteins have never been described in the context of MFM so far. A subset of proteins including desmin, filamin C, Xin, Xirp2 and many other proteins was detected in aggregates in all patients. The comparison of MFM subtypes revealed disease-specific patterns of aggregate compositions with clear differences in ratios of most abundant proteins. Filamin C, desmin, myotilin and ZASP showed the highest accumulation in aggregates of related MFM subtypes. Our proteomic data provide essential new insights in the composition of pathological protein aggregates in MFM. Proteomic profiles of aggregates seem to be specific for the different MFM subtypes and expand our knowledge about proteins involved in pathogenesis of filaminopathy, desminopathy, myotilinopathy and ZASPopathy. The list of abundant proteins in aggregates also include potential new MFM proteins. These should to be considered in future genetic studies in MFM patients with so far unknown mutation.