DNM2 mutations in a cohort of sporadic patients with centronuclear myopathy

Centronuclear myopathy (CNM) is a rare congenital muscle disease characterized by fibers with prominent centralized nuclei in muscle biopsies. The disease is clinically heterogeneous, ranging from severe neonatal hypotonic phenotypes to adult-onset mild muscle weakness, and can have multiple modes of inheritance in association with various genes, including MTM1, DNM2, BIN1 and RYR1. Here we analyzed 18 sporadic patients with clinical and histological diagnosis of CNM and sequenced the DNM2 gene, which codes for the dynamin 2 protein. We found DNM2 missense mutations in two patients, both in exon 8, one known (p.E368K) and one novel (p.F372C), which is found in a position of presumed pathogenicity and appeared de novo. The patients had similar phenotypes characterized by neonatal signs followed by improvement and late childhood reemergence of slowly progressive generalized muscle weakness, elongated face with ptosis and ophthalmoparesis, and histology showing fibers with radiating sarcoplasmic strands (RSS). These patients were the only ones in the series to present this histological marker, which together with previous reports in the literature suggest that, when RSS are present, direct sequencing of DNM2 mutation hot spot regions should be the first step in the molecular diagnosis of CNM, even in sporadic cases.     

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.     

Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.     

Centronuclear Myopathy in Labrador Retrievers: A Recent Founder Mutation in the PTPLA Gene Has Rapidly Disseminated Worldwide

Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ∼70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLA^cnm mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLA^cnm carriers in 13 countries. Haplotype analysis demonstrated that the PTPLA^cnm allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM.     

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna^H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna^H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.     

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.     

Gain-of-Function Mutations in RIT1 Cause Noonan Syndrome,a RAS/MAPK Pathway Syndrome

RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes.     

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM^∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.     

Elevated MTORC1 signaling and impaired autophagy

A-type lamins, generated from the LMNA gene by differential splicing, are type V intermediate filament proteins that polymerize to form part of the nuclear lamina, and are of considerable medical interest because missense mutations in LMNA give rise to a wide range of dystrophic and progeroid syndromes. Among these are dilated cardiomyopathy and two forms of muscular dystrophy (limb-girdle and Emery-Dreifuss), which are modeled in lmna^?/? mice and mice engineered to express human disease mutations. Our recent study demonstrates that cardiac and skeletal muscle pathology in lmna^?/? mice can be attributed to elevated MTORC1 signaling leading to impairment of autophagic flux. An accompanying paper from another laboratory shows similar impairments in mice engineered to express the LMNA H222P associated with dilated cardiomyopathy in humans and also in left ventricular tissue from human subjects. MTORC1 inhibition with rapalogs restores autophagic flux and improves cardiac function in both mouse models, and extends survival in the lmna^?/? mice. These findings elaborate a potential treatment option for dilated cardiomyopathy and muscular dystrophy associated with LMNA mutation and supplement growing evidence linking impaired autophagy to human disease.     

OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency

Background

Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.

Results

We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.

Conclusions

OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.     

Role of troponin T in disease

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease. (Mol Cell Biochem 263: 115–129, 2004)     

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.     

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

Background

Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna^H222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna^H222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit.

Methods

Male Lmna^H222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated.

Results

Treatment of Lmna^H222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional shortening at 20 weeks of age.

Conclusions

Both ACE inhibition and MEK1/2 inhibition have beneficial effects on left ventricular function in Lmna^H222P/H222P mice and both drugs together have a synergistic benefit when initiated after the onset of left ventricular dysfunction. These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor in addition to standard of care in patients with dilated cardiomyopathy caused by LMNA mutations.     

Troponin and cardiomyopathy

The troponin complex was discovered over thirty years ago and since then much insight has been gained into how this complex senses fluctuating levels of Ca²⁺ and transmits this signal to the myofilament. Advances in genetics methods have allowed identification of mutations that lead to the phenotypically distinct cardiomyopathies: hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and dilated cardiomyopathy (DCM). This review serves to highlight key in vivo studies of mutation effects that have followed many years of functional studies and discusses how these mutations alter energetics and promote the characteristic remodeling associated with cardiomyopathic diseases. Studies have been performed that examine alterations in signaling and genomic methods have been employed to isolate upregulated proteins, however these processes are complex as there are multiple roads to hypertrophy or dilation associated with genetic cardiomyopathies. This review suggests future directions to explore in the troponin field that would heighten our understanding of the complex regulation of cardiac muscle contraction.     

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.     

The<Emphasis Type="Italic"> cnm</Emphasis> locus,a canine homologue of human autosomal forms of centronuclear myopathy,maps to chromosome 2

Myotubular/centronuclear myopathies are a nosological group of hereditary disorders characterised by severe architectural and metabolic remodelling of skeletal muscle fibres. In most myofibres, nuclei are found at an abnormal central position within a halo devoid of myofibrillar proteins. The X-linked form (myotubular myopathy) is the most prevalent and severe form in human, leading to death during early postnatal life. Maturation of fibres is not completed and fibres resemble myotubes. Linkage analysis in human has helped to identify MTM1 as the morbid gene. MTM1 encodes myotubularin, a dual protein phosphatase. In families in which myotubular myopathy segregates, detected mutations in MTM1 abolish the specific phosphatase activity targeting the second messenger phosphatidylinositol 3-phosphate. Autosomal forms (centronuclear) have a later onset and are often compatible with life. At birth, fibres are normally constituted but progressively follow remodelling with a secondary centralisation of nuclei. Their prevalence is low; hence, no linkage data can be performed and no molecular aetiology is known. In the Labrador Retriever, a spontaneous disorder strikingly mimics the clinical evolution of the human centronuclear myopathy. We have established a canine pedigree and show that the disorder segregates as an autosomal recessive trait in that pedigree. We have further mapped the dog locus to a region on chromosome 2 that is orthologous to human chromosome 10p. To date, no human MTM1 gene member has been mapped to this genetic region. This report thus describes the first spontaneous mammalian model of centronuclear myopathy and defines a new locus for this group of diseases.