Postural Muscle Atrophy Research Articles

ВЛИЯНИЕ ИНГИБИТОРА КИСЛОЙ СФИНГОМИЕЛИНАЗЫ НА РЕГУЛЯЦИЮ ЭКСПРЕССИИ БЫСТРЫХ И МЕДЛЕННЫХ ИЗОФОРМ ТЯЖЕЛЫХ ЦЕПЕЙ МИОЗИНА В КАМБАЛОВИДНОЙ МЫШЦЕ КРЫС ПРИ 7-СУТОЧНОЙ ФУНКЦИОНАЛЬНОЙ РАЗГРУЗКЕ

Functional unloading of postural muscles leads to atrophy and changes in regulation of myosin heavy chains (MyHC) expression resulting in shifting the muscle phenotype towards a more active synthesis of fast MHCs isoforms. This investigation was concerned with regulation of Iβ, IIA, IIB and IIDX expression in rat m. soleus after 7-day tail-suspension; some of the suspended rats were treated with amitriptyline, an acid sphingomyelinase (ASM) inhibitor. Expression of the MyHC genes was assessed using real-time PCR and western-blotting. Blotting was also used to determine levels of phosphorylated and unphosphorylated forms of proteins involved in the regulation of muscle myosin phenotype (NFAT1 and MyoD1). Immunofluorescence microscopy was used to analyze muscle sections for changes in ceramide and ASM, and morphological signs of atrophy. It was shown that amitryptiline prevented such typical unloading-induced changes in m. soleus phenotype, as a decreased expression of slow MyHC isoform and increased expression of fast isoforms. Also, amitryptiline prevented muscle atrophy, activation of MyoD1 and inactivation of NFAT1. These observations indicate participation of ASM and ceramide in signaling pathways involved in the development of postural muscle atrophy and changes in its myosin phenotype caused by functional unloading.

Comparison of trunk muscle exercises in supine position during short arm centrifugation with 1g at centre of mass and upright in 1g.

Spaceflight is associated with reduced antigravitational muscle activity, which results in trunk muscle atrophy and may contribute to post-flight postural and spinal instability. Exercise in artificial gravity (AG) performed via short-arm human centrifugation (SAHC) is a promising multi-organ countermeasure, especially to mitigate microgravity-induced postural muscle atrophy. Here, we compared trunk muscular activity (mm. rectus abdominis, ext. obliques and multifidi), cardiovascular response and tolerability of trunk muscle exercises performed during centrifugation with 1 g at individual center of mass on a SAHC against standard upright exercising. We recorded heart rate, blood pressure, surface trunk muscle activity, motion sickness and rating of perceived exertion (BORG) of 12 participants (8 male/4 female, 34 ± 7 years, 178.4 ± 8.2 cm, 72.1 ± 9.6 kg). Heart rate was significantly increased (p < 0.001) during exercises without differences in conditions. Systolic blood pressure was higher (p < 0.001) during centrifugation with a delayed rise during exercises in upright condition. Diastolic blood pressure was lower in upright (p = 0.018) compared to counter-clockwise but not to clockwise centrifugation. Target muscle activation were comparable between conditions, although activity of multifidi was lower (clockwise: p = 0.003, counter-clockwise: p < 0.001) and rectus abdominis were higher (clockwise: p = 0.0023, counter-clockwise: < 0.001) during centrifugation in one exercise type. No sessions were terminated, BORG scoring reflected a relevant training intensity and no significant increase in motion sickness was reported during centrifugation. Thus, exercising trunk muscles during centrifugation generates comparable targeted muscular and heart rate response and appears to be well tolerated. Differences in blood pressure were relatively minor and not indicative of haemodynamic challenge. SAHC-based muscle training is a candidate to reduce microgravity-induced inter-vertebral disc pathology and trunk muscle atrophy. However, further optimization is required prior to performance of a training study for individuals with trunk muscle atrophy/dysfunction.

P.053 A clinical mystery finally revealed

Background: We report 2 brothers sharing FHL1 identified mutation. They presented in childhood with overlapping clinical features characterized by early onset stiffness and increased muscle definition with cardiac involvement. After 30 years of neurological followup, the diagnosis is finally revealed. Methods: At early ages, both had increased definition of upper trunk musculature. The older brother had hypophonic voice with raspy character, which is to our knowledge, not reported with this mutation before. He required a pacemaker for arrhythmias, while the younger developed congestive heart failure. Results: Their initial investigations failed to unveil a diagnosis, including a negative next generation sequencing (NGS) panel for AR LGMD. An expanded NGS sent on the older brother revealed he is hemizygous for 1770 bp deletion within FHL 1 gene, this deletion includes exon 7to 8, and confirmed on the other. Conclusions: First reported in 2008, FHL1 mutations result in phenotypically distinct neuromuscular disorders: X-linked myopathy with Postural Muscle Atrophy and generalized hypertrophy, X-linked dominant scapuloperoneal Myopathy, and Reducing Body Myopathy. Subsequently other phenotypes have been reported including Emery-Dreifuss muscular dystrophy and hypertrophic cardiomyopathy. Our patients present with a phenotype that had been reported with FHL1 mutation, highlighting the possible recognition of this presentation in aiding a diagnostic approach.

FHL1-related myopathy may not be classified by reducing bodies in muscle biopsy

FHL1-related myopathies, including reducing body myopathy (RBM), X-linked scapulo-axio-peroneal myopathy, rigid spine syndrome, X-linked myopathy with postural muscle atrophy (XMPMA), X-linked Emery-Dreifuss muscular dystrophy and hypertrophic cardiomyopathy, are clinically and pathologically heterogeneous disorders caused by FHL1 gene mutations. According to previous reports, the first three types are myopathies with reducing bodies observed in biopsies, and the last three are myopathies without reducing bodies. We report four FHL1-related myopathy patients, including an XMPMA patient and a RBM family with three patients. Clinical information, muscle biopsies, electromyograms and genetic testing were obtained. Muscle weakness and atrophy, spinal rigidity, and joint contracture were present in the RBM family. The XMPMA patient showed a pseudoathletic appearance with muscle weakness and atrophy, spinal rigidity and deformity. The index patient of the RBM family underwent two muscle biopsies to find reducing bodies. Interestingly, these muscle biopsies revealed reducing bodies and rimmed vacuoles not only in the RBM family but also in the XMPMA patient. Next-generation sequencing identified a reported single missense mutation c.448 C>T (p. C150R) in the RBM family and a novel mutation c.814T>C (p. S272P) in the XMPMA patient. Therefore, FHL1-related myopathies overlap substantially and may not be simply classified into subtypes depending on reducing bodies. Biopsies of additional affected muscles can aid in finding reducing bodies. We report the first XMPMA patient with a novel FHL1 mutation and reducing bodies in a muscle biopsy in China.

AMP-Activated Protein Kinase as a Key Trigger for the Disuse-Induced Skeletal Muscle Remodeling

Molecular mechanisms that trigger disuse-induced postural muscle atrophy as well as myosin phenotype transformations are poorly studied. This review will summarize the impact of 5′ adenosine monophosphate -activated protein kinase (AMPK) activity on mammalian target of rapamycin complex 1 (mTORC1)-signaling, nuclear-cytoplasmic traffic of class IIa histone deacetylases (HDAC), and myosin heavy chain gene expression in mammalian postural muscles (mainly, soleus muscle) under disuse conditions, i.e., withdrawal of weight-bearing from ankle extensors. Based on the current literature and the authors’ own experimental data, the present review points out that AMPK plays a key role in the regulation of signaling pathways that determine metabolic, structural, and functional alternations in skeletal muscle fibers under disuse.

Fhl1 W122S causes loss of protein function and late-onset mild myopathy.

A member of the four-and-a-half-LIM (FHL) domain protein family, FHL1, is highly expressed in human adult skeletal and cardiac muscle. Mutations in FHL1 have been associated with diverse X-linked muscle diseases: scapuloperoneal (SP) myopathy, reducing body myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS) and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and investigated the effects of this mutation at three time points (3-5 months, 7-10 months and 18-20 months) in hemizygous male and heterozygous female mice. Survival was comparable in mutant and wild-type animals. We observed decreased forelimb strength and exercise capacity in adult hemizygous male mice starting from 7 to 10 months of age. Western blot analysis showed absence of Fhl1 in muscle at later stages. Thus, adult hemizygous male, but not heterozygous female, mice showed a slowly progressive phenotype similar to human patients with late-onset muscle weakness. In contrast to SP myopathy patients with the FHL1 W122S mutation, mutant mice did not manifest cytoplasmic inclusions (reducing bodies) in muscle. Because muscle weakness was evident prior to loss of Fhl1 protein and without reducing bodies, our findings indicate that loss of function is responsible for the myopathy in the Fhl1 W122S knock-in mice.

G.P.150: Clinical heterogeneity in adult forms of FHL1 related myopathies. The “Institut de Myologie” experience

FHL1 gene mutations are responsible for reducing body myopathy (RBM), a rare condition characterized by progressive muscle weakness and the presence of intracytoplasmic aggregates. Age at onset ranges from early onset in infancy, through childhood and in some cases adult age. FHL1 mutations may also lead to allelic disorders including Emery-Dreifuss like muscular dystrophy (EDMD), hypertrophic cardiomyopathy (HCM), X-linked myopathy with postural muscle atrophy and generalized hypertrophy (X-MPMA) and X-linked scapuloperoneal myopathy (X-SM). To report clinical, muscle imaging, histological and genetic features found of adult patients carrying FHL1 mutations, we retrospectively reviewed their medical reports of the 11 patients (6 M, 5 F) belonging to 8 families followed at the “Institute de Myologie”, Pitie-Salpetriere hospital, Paris. The 11 patients were assessed from 13 to 52 years old. Pseudodominant mode of inheritance was suggested in 4 families, X-linked in 3 and sporadic in one. Four had EDMD, 2 X-SM, 1 X-MPMA, 1 HCM while 2 patients had atypical features and 1 was still asymptomatic. When symptomatic, female patients had a striking asymmetric muscle involvement. Five patients had hypertrophic cardiomyopathy requiring heart transplantation in one patient at 18 years old. Two patients required night time ventilation due to diaphragm paralysis. Histologically, only 3 patients among the 8 probands had reducing bodies (RBs) at muscle biopsy, while 3 had non specific pattern, 1 had neuropathic pattern and one muscle biopsy was considered as normal. In contrast to infantile forms of FHL1 related myopathy that usually show RBs at muscle biopsy and lead to early death, adult forms had wider clinical and histological presentations. Moreover, FHL1 gene screening should be considered in those undiagnosed patients suffering from myopathies with diaphragm paralysis and/or HCM where X-linked inheritance is not excluded even in the absence of RBs at muscle pathology.

FHL1 mutations that cause clinically distinct human myopathies form protein aggregates and impair myoblast differentiation

FHL1 mutations cause several clinically heterogeneous myopathies, including reducing body myopathy (RBM), scapuloperoneal myopathy (SPM) and X-linked myopathy with postural muscle atrophy (XMPMA). The molecular mechanisms underlying the pathogenesis of FHL1 myopathies are unknown. Protein aggregates, designated 'reducing bodies', that contain mutant FHL1 are detected in RBM muscle but not in several other FHL1 myopathies. Here, RBM, SPM and XMPMA FHL1 mutants were expressed in C2C12 cells and showed equivalent protein expression to wild-type FHL1. These mutants formed aggregates that were positive for the reducing body stain Menadione-NBT, analogous to RBM muscle aggregates. However, hypertrophic cardiomyopathy (HCM) and Emery-Dreifuss muscular dystrophy (EDMD) FHL1 mutants generally exhibited reduced expression. Wild-type FHL1 promotes myoblast differentiation; however, RBM, SPM and XMPMA mutations impaired differentiation, consistent with a loss of normal FHL1 function. Furthermore, SPM and XMPMA FHL1 mutants retarded myotube formation relative to vector control, consistent with a dominant-negative or toxic function. Mutant FHL1 myotube formation was partially rescued by expression of a constitutively active FHL1-binding partner, NFATc1. This is the first study to show that FHL1 mutations identified in several clinically distinct myopathies lead to similar protein aggregation and impair myotube formation, suggesting a common pathogenic mechanism despite heterogeneous clinical features.

P.5.19 Fhl1 W122S knock-in mice manifest late-onset mild myopathy

FHL1 is a member of the four-and-a half-LIM (FHL) domain protein family and it is highly expressed in adult human skeletal muscle. It is believed to participate in sarcomere assembly, muscle growth and differentiation, and in the biomechanical stress responses. Mutations in FHL1 gene have been associated with different myopathies, including reducing body myopathy, scapuloperoneal (SP) myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We have generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and have investigated the effects of this mutation at pre-symptomatic, phenotypic-onset, and late-stage of the disease, in hemizygous males and heterozygous females mice. Survival was comparable in mutant and wild-type animals. Adult hemizygous males mice showed a slowly progressive phenotype similar to human patients with relatively late-onset muscle weakness. In particular, we observed reduced forelimb strength and exercise capacity. Western blot analysis showed absence of FHL1 protein in muscle of hemizygous males at advanced stages. This animal model may help to elucidate the role of FHL1 in skeletal muscle and the pathomechanism of FHL1 mutations in X-linked dominant scapuloperoneal myopathy.

G.P.121 X-linked recessive distal myopathy with hypertrophic cardiomyopathy caused by a novel mutation in the FHL1 gene

<h2>Abstract</h2> The four and a half LIM domain protein 1 (<i>FHL1</i>) gene encodes FHL1, a LIM domain containing protein that regulates skeletal and cardiac muscle function. <i>FHL1</i> mutations are associated with variable phenotypes including reducing body myopathy, X-linked myopathy with postural muscle atrophy, scapuloperoneal myopathy and Emery–Dreifuss muscular dystrophy. The <i>FHL1</i> gene encodes three FHL1 isoforms (FHL1A, FHL1B, FHL1C). Patients with <i>FHL1</i> mutations affecting all three isoforms exhibit a more severe phenotype, whereas mutations that affect only FHL1A are less severe. We describe a boy with a family history consistent with X-linked distal myopathy/cardiomyopathy. The boy first presented at age 8 with exercise intolerance and was found to have pes cavus, distal wasting, weakness of ankle dorsiflexion and areflexia. There was no clinical evidence of cardiac involvement. The patient's brother had died suddenly from isolated hypertrophic cardiomyopathy some years prior. A maternal uncle had also died suddenly from complications of cardiomyopathy. In this subject, an echocardiogram at age 8 was normal, but by age 14 there was evidence of a hypertrophic cardiomyopathy. Muscle biopsy showed myopathic changes and prominent rimmed vacuoles. Sequencing of the <i>FHL1</i> gene revealed a novel hemizygous c.764G>C missense mutation in exon 8, causing a C255S substitution affecting one of the zinc binding cysteine residues in the fourth LIM domain of FHL1A. The patient's mother and maternal aunt were heterozygous for the mutation. No suitable samples could be identified for testing of the deceased sibling and maternal uncle. Over 26 <i>FHL1</i> mutations have been reported to date, many of which cause phenotypes with shared clinicopathological features. This first report of a predominantly distal myopathy with hypertrophic cardiomyopathy occurring secondary to an <i>FHL1</i> mutation further expands the clinical spectrum of <i>FHL1</i>-related myopathies.

G.P.80 Non-sense mutation in exon two of the dystrophin gene results in mild Becker muscular dystrophy with generalized muscle hypertrophy

Abstract Becker muscular dystrophy leads to progressive proximal weakness. Focal wasting is commonly seen in the calves, less typically in other locations such as the thenar eminence. Generalized muscle hypertrophy has been described with other muscular dystrophies such as LGMD 1A and in a X-linked myopathy with postural muscle atrophy caused by mutation in the FHL gene. Generalized muscle hypertrophy with Becker muscular dystrophy (BMD) has not been reported before. We present a patient with muscle pain from adolescence. On examination he had generalized muscle hypertrophy, but preserved muscle strength and a 10- to 20-fold elevated CPK. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. MRI showed a normal distribution between muscle and fat confirming that his large body volume was caused by generalized muscle hypertrophy. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with generalized muscle hypertrophy. The unusual clinical presentation suggests that the phenotype is caused by a translation re-initiation downstream for the mutation site.

EMERY-DREIFUSS MUSCULAR DYSTROPHY AND FHL-1 RELATED CONDITIONS - POSTER PRESENTATIONS G.P.119 How mutations in the FHL1 gene lead to severe muscle disease

<h2>Abstract</h2> The LIM domain containing protein FHL1 promotes skeletal muscle growth however mutations in <i>FHL1</i> gene cause four different muscle diseases: Reducing Body Myopathy (RBM), Scapuloperoneal Myopathy (SPM), X-Linked Myopathy with Postural Muscle Atrophy (XMPMA) and Emery-Dreifuss Muscular Dystrophy (EDMD). Protein aggregation and childhood lethality occur in RBM, while XMPMA and EDMD exhibit adult onset with no protein aggregation. How FHL1 mutations lead to muscle disease and why severity differs between these diseases is unknown. We hypothesised that FHL1 mutations disrupt key structural residues, leading to miss-folding and the formation of reducing bodies which are unique to RBM. Aggregation is hypothesised to be toxic and may cause the loss of normal FHL1 function during muscle differentiation. To determine how FHL1 mutations lead to aggregation and cause myopathy, different FHL1 mutants representative of each disease and mutation type were over-expressed in C2C12 myoblasts as they differentiated to form myotubes. Protein aggregation was assessed by immunofluorescence and Western blot and of FHL1 together with markers for protein aggregation and Menadione-NBT staining for reducing bodies, which are clinically specific to RBM. Myotube differentiation was assessed by immunofluorescence and Western blot of myogenin and myosin heavy chain (MHC). Although protein aggregation is only clinically associated with RBM we show other FHL1 mutations reported in SPM and XMPMA also lead to aggregate formation in C2C12 myotubes in a time dependant manor. These aggregates contained markers observed in RBM patient biopsies including ubiquitin and GRP78, and were positive for Menadione-NBT staining. Wild-type FHL1 did not aggregate and had a similar level of expression compared to the FHL1 mutants. This study suggests that aggregation may be a common hallmark of FHL1 myopathies and that these diseases may potentially share some common pathological mechanisms.

Spongious Hypertrophic Cardiomyopathy in Patients With Mutations in the Four-and-a-Half LIM Domain 1 Gene

X-linked myopathy with postural muscle atrophy is a novel X-linked myopathy caused by mutations in the four-and-a-half LIM domain 1 gene (FHL1). Cardiac involvement was suspected in initial publications. We now systematically analyzed the association of the FHL1 genotype with the cardiac phenotype to establish a potential cardiac involvement in the disease. Seventeen male patients and 23 female mutation carriers were compared with healthy controls. Every patient underwent a comprehensive clinical and cardiovascular workup. ECG abnormalities occurred frequently in affected males and were less frequent in heterozygous females. Both male and female mutation carriers had increased myocardial mass (affected males=115.1±25.3 g/m(2); heterozygous females=95.1±19.6 g/m(2); controls=89.0±15.6 g/m(2) and 72.6±12.6 g/m(2); respectively) with increased wall thickness (typically midventricular and apical segments) mainly in affected males. Longitudinal systolic function was reduced in affected males (radial systolic strain: affected males=24.6±11.8%; male controls=43.2±14.8%; P=0.002). Diastolic dysfunction occurred in both affected males and heterozygous females. Cardiac MRI revealed a morphological hallmark of X-linked myopathy with postural muscle atrophy; a characteristic spongious structure and replacement fibrosis indicated by late enhancement could be detected in most affected males. X-linked myopathy with postural muscle atrophy was associated with reduced exercise capacity in affected males but not in heterozygous female mutation carriers. X-linked myopathy with postural muscle atrophy patients consistently showed electrical, functional, and characteristic morphological cardiac abnormalities that translate into reduced exercise capacity. Reduced systolic and diastolic function is associated with a novel type of spongious hypertrophic cardiomyopathy. An unexpected finding was that some cardiac abnormalities were also present in heterozygous female mutation carriers.

Reducing Body Myopathy and Other FHL1-Related Muscular Disorders

During the past 2 years, considerable progress in the field of four and a half LIM domain protein 1 (FHL1)-related myopathies has led to the identification of a growing number of FHL1 mutations. This genetic progress has uncovered crucial pathophysiological concepts, thus redefining clinical phenotypes. Important new characterizations include 4 distinct human myopathies: reducing body myopathy, X-linked myopathy with postural muscle atrophy, Emery-Dreifuss muscular dystrophy, and scapuloperoneal myopathy. Additionally, FHL1 mutations have been discovered in rigid spine syndrome and in a single family with contractures, rigid spine, and cardiomyopathy. In this review, we focus on the clinical phenotypes, which we correlate with the novel genetic and histological findings encountered within FHL1-related myopathies. This correlation will frequently lead to a considerably expanded clinical spectrum associated with a given FHL1 mutation.

XMPMA: acute on chronic ventilatory failure managed successfully with non-invasive ventilation

X-linked myopathy with postural muscle atrophy (XMPMA) is a recently described myopathy; it may cause symptomatic sleep disordered breathing which can be managed with non-invasive ventilation.

Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel Kv1.5

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G0/G1 phase. Furthermore, low expression of Kv1.5, a voltage-gated potassium channel known to alter myoblast proliferation during the G1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between Kv1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and Kv1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of Kv1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K+ currents when compared to oocytes expressing Kv1.5 only. We here present the first evidence on a biological relevance of FHL1C.

Christianson syndrome in a patient with an interstitial Xq26.3 deletion

Interstitial deletions of chromosome band Xq26.3 are rare. We report on a 2-year-old boy in whom array comparative genomic hybridization analysis revealed an interstitial 314 kb deletion in Xq26.3 affecting SLC9A6 and FHL1. Mutations in SLC9A6 are associated with Christianson syndrome (OMIM 300243), a syndromic form of X-linked mental retardation (XLMR) characterized by microcephaly, severe global developmental delay, ataxia and seizures. FHL1 mutations cause Emery-Dreifuss muscular dystrophy (OMIM 310300), X-linked myopathy with postural muscle atrophy (XMPMA, OMIM 300696), scapuloperoneal myopathy (OMIM 300695), or reducing body myopathy (OMIM 300717, 300718). The clinical problems of the patient reported here comprised severe intellectual disability, absent speech, ataxia, epilepsy, and gastroesophageal reflux, and could mostly be attributed to SLC9A6 insufficiency. In contrast to the majority of reported Christianson syndrome patients who were microcephalic, this patient was normocephalic, but his head circumference had decelerated from the 50th centile at birth to the 25th centile at the age of 2 ²/¹² years. Muscle problems due to the FHL1 deletion are not to be expected before late childhood, which is the earliest age of onset for FHL1 associated Emery-Dreifuss muscular dystrophy. This patient broadens the spectrum of SLC9A6 mutations and contributes to the clinical delineation of Christianson syndrome. This is also the first patient with a deletion affecting both SLC9A6 and the complete FHL1 gene.

Phenotypic heterogeneity in British patients with a founder mutation in the FHL1 gene

Mutations in the four-and-a-half LIM domain 1 (FHL1) gene, which encodes a 280-amino-acid protein containing four LIM domains and a single zinc-finger domain in the N-terminal region, have been associated with a broad clinical spectrum of X-linked muscle diseases encompassing a variety of different phenotypes. Patients might present with a scapuloperoneal myopathy, a myopathy with postural muscle atrophy and generalized hypertrophy, an Emery-Dreifuss muscular dystrophy, or an early onset myopathy with reducing bodies. It has been proposed that the phenotypic variability is related to the position of the mutation within the FHL1 gene. Here, we report on three British families with a heterogeneous clinical presentation segregating a single FHL1 gene mutation and haplotype, suggesting that this represents a founder mutation. The underlying FHL1 gene mutation was detected by direct sequencing and the founder effect was verified by haplotype analysis of the FHL1 gene locus. A 3-bp insertion mutation (p.Phe127_Thr128insIle) within the second LIM domain of the FHL1 gene was identified in all available affected family members of the three families. Haplotype analysis of the FHL1 region on Xq26 revealed that the families shared a common haplotype. The p.Phe127_Thr128insIle mutation in the FHL1 gene therefore appears to be a British founder mutation and FHL1 gene screening, in particular of exon 6, should therefore be indicated in British patients with a broad phenotypic spectrum of X-linked muscle diseases.

Four and a Half Lim Domains (FHL) Genes Reduce Conductivity of the KCNA5 Channel

Myopathies are inherited muscle disorders characterized by weakness and atrophy of voluntary skeletal muscles, sometimes including the cardiac muscle. A phenotypically distinct, X-linked myopathy with postural muscle atrophy, termed XMPMA, has been recently described and linked to mutations in the FHL1 gene. FHL1, a member of LIM-only proteins, is expressed in skeletal and cardiac muscle and suggested to play a role in sarcomere synthesis and assembly. Three splice variants (A, B and C) exist, which differ in expression pattern, binding partners and subcellular localization. A mutation found in a large XMPMA family (C224W) affects only isoforms A and B.Aim of our study is to functionally characterize mutated FHL1 isoforms and their interaction with the voltage-gated potassium channel (KCNA5 or Kv1.5), which is involved in cardiac excitability. These interactions may partly explain the cardiac involvement within the clinical spectrum of XMPMA patients.K+ currents were recorded in Xenopus leavis oocytes injected with KCNA5 mRNA with or without coexpression of FHL1AWT, FHL1AC224W or FHL1C. Upon coexpression of all three FHL1 proteins, K+ current density was differently decreased, when compared to oocytes expressing KCNA5 alone. Kinetics of the channel was not affected. These results support the role of FHL1 as a key molecular component in regulation of expression of KCNA5. Future experiments will concentrate on colocalization and molecular interaction of FHL1 and KCNA5 in mammalian in vitro systems (HL1 cells).Support by Graz Medical University Ph.D. programme and County of Styria (GZ: A3-16.R-10/2009-112).

SLIMMER (FHL1B/KyoT3) Interacts with the Proapoptotic Protein Siva-1 (CD27BP) and Delays Skeletal Myoblast Apoptosis

The fhl1 gene encoding four-and-a-half LIM protein-1 (FHL1) and its spliced isoform, SLIMMER, is mutated in reducing body myopathy, X-linked myopathy with postural muscle atrophy, scapuloperoneal myopathy, and rigid spine syndrome. In this study we have identified a novel function for SLIMMER in delaying skeletal muscle apoptosis via an interaction with the proapoptotic protein Siva-1. Siva-1 was identified as a SLIMMER-specific-interacting protein using yeast two-hybrid screening, direct-binding studies, and glutathione S-transferase pulldown analysis of murine skeletal muscle lysates. In C2C12 skeletal myoblasts, SLIMMER and Siva co-localized in the nucleus; however, both proteins exhibited redistribution to the cytoplasm following the differentiation of mononucleated myoblasts to multinucleated myotubes. In sections of mature skeletal muscle from wild type mice, SLIMMER and Siva-1 co-localized at the Z-line. SLIMMER and Siva-1 were also enriched in Pax-7-positive satellite cells, muscle stem cells that facilitate repair and regeneration. Significantly, SLIMMER delayed Siva-1-dependent apoptosis in C2C12 myoblasts. In skeletal muscle sections from the mdx mouse model of Duchenne muscular dystrophy, SLIMMER and Siva-1 co-localized in the nucleus of apoptotic myofibers. Therefore, SLIMMER may protect skeletal muscle from apoptosis.