Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His₆ tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μm) is ∼ 4-fold weaker than for β-S1 (25 μm). Correspondingly, the MgADP release rate for α-S1 (350 s⁻¹) is ∼3-fold greater than for β-S1 (120 s⁻¹). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s⁻¹). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.     

Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.     

Genotype-phenotype correlation in Japanese patients with familial Mediterranean fever: differences in genotype and clinical features between Japanese and Mediterranean populations

Introduction

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent self-limiting fever and serositis that mainly affects Mediterranean populations. Many patients with FMF have been reported in Japan due to increasing recognition of this condition and the availability of genetic analysis for the gene responsible, MEFV. The present study was performed to elucidate the clinical characteristics of Japanese FMF patients and to examine the precise genotype-phenotype correlation in a large cohort of Japanese FMF patients.

Methods

We analyzed the MEFV genotypes and clinical manifestations in 116 patients clinically diagnosed as having FMF and with at least one mutation.

Results

The most frequent mutation in Japanese patients was E148Q (40.2%), followed by M694I (21.0%), L110P (18.8%), P369S (5.4%), and R408Q (5.4%). In contrast, common mutations seen in Mediterranean patients, such as M694V, V726A, and M680I, were not detected in this population. The clinical features with M694I were associated with more severe clinical course compared to those seen with E148Q. P369S/R408Q showed variable phenotypes with regard to both clinical manifestations and severity. Patients with M694I showed a very favorable response to colchicine therapy, while those with P369S and R408Q did not.

Conclusions

Clinical features and efficacy of treatment in Japanese FMF patients vary widely according to the specific MEFV gene mutation, and therefore genetic analysis should be performed for diagnosis in cases of Japanese FMF.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0439-7) contains supplementary material, which is available to authorized users.     

Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

Background

Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins.

Principal Findings

Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo.

Significance

These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.     

Electron Tomography of Cryofixed,Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Background

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology

We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.

Conclusion

We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.     

ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2

Background

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson''s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro.

Methodology/Principal Findings

Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7.

Conclusions/Significance

Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson''s disease pathogenesis.     

Involvement of the Same TNFR1 Residue in Mendelian and Multifactorial Inflammatory Disorders

Objectives

TNFRSF1A is involved in an autosomal dominant autoinflammatory disorder called TNFR-associated periodic syndrome (TRAPS). Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable. This is especially true for the frequent R92Q mutation, which might not be responsible for TRAPS per se but represents a susceptibility factor to multifactorial inflammatory disorders. This study investigates TRAPS pathophysiology in a family exceptional by its size (13 members) and compares the consequences of several mutations affecting arginine 92.

Methods

TNFRSF1A screening was performed by PCR-sequencing. Comparison of the 3-dimensional structure and electrostatic properties of wild-type and mutated TNFR1 proteins was performed by in silico homology modeling. TNFR1 expression was assessed by FACS analysis, western blotting and ELISA in lysates and supernatants of HEK293T cells transiently expressing wild-type and mutated TNFR1.

Results

A TNFRSF1A heterozygous missense mutation, R92W (c.361C>T), was shown to perfectly segregate with typical TRAPS manifestations within the family investigated (p<5.10⁻⁴). It was associated with very high disease penetrance (0.9). Prediction of its impact on the protein structure revealed local conformational changes and alterations of the receptor electrostatic properties. R92W also impairs the TNFR1 expression at the cell surface and the levels of soluble receptor. Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity. In contrast, TNFR1-R92Q behaves like the wild-type receptor.

Conclusions

These data demonstrate the pathogenicity of a mutation affecting arginine 92, a residue whose involvement in inflammatory disorders is deeply debated. Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

Differential cross-bridge kinetics of FHC myosin mutations R403Q and R453C in heterozygous mouse myocardium

The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from heterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament (V(RTF)) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal V(RTF) was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production (b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (pCa(50) 5.64 +/- 0.02) and significantly enhanced in R453C/+ (5.82 +/- 0.03) compared with WT (5.58 +/- 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level.     

Participation of myosin Va and Pka type I in the regeneration of neuromuscular junctions

Background

The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration.

Methodology/Principal Findings

To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology.

Conclusions/Significance

Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle.     

Elevated Heart Rate Triggers Action Potential Alternans and Sudden Death. Translational Study of a Homozygous KCNH2 Mutation

Background

Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed “human HERG knockout” and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness.

Methods and Results

Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins – both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V₅₀ in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q.

Conclusion

Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.     

Functional Consequences of a Novel Variant of PCSK1

Background

Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.

Methodology/Principal Findings

A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.

Conclusion/Significance

Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.     

Exercise Training Reverses Skeletal Muscle Atrophy in an Experimental Model of VCP Disease

Background

The therapeutic effects of exercise resistance and endurance training in the alleviation of muscle hypertrophy/atrophy should be considered in the management of patients with advanced neuromuscular diseases. Patients with progressive neuromuscular diseases often experience muscle weakness, which negatively impact independence and quality of life levels. Mutations in the valosin containing protein (VCP) gene lead to Inclusion body myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD) and more recently affect 2% of amyotrophic lateral sclerosis (ALS)-diagnosed cases.

Methods/Principle Findings

The present investigation was undertaken to examine the effects of uphill and downhill exercise training on muscle histopathology and the autophagy cascade in an experimental VCP mouse model carrying the R155H mutation. Progressive uphill exercise in VCP^R155H/+ mice revealed significant improvement in muscle strength and performance by grip strength and Rotarod analyses when compared to the sedentary mice. In contrast, mice exercised to run downhill did not show any significant improvement. Histologically, the uphill exercised VCP^R155H/+ mice displayed an improvement in muscle atrophy, and decreased expression levels of ubiquitin, P62/SQSTM1, LC3I/II, and TDP-43 autophagy markers, suggesting an alleviation of disease-induced myopathy phenotypes. There was also an improvement in the Paget-like phenotype.

Conclusions

Collectively, our data highlights that uphill exercise training in VCP^R155H/+ mice did not have any detrimental value to the function of muscle, and may offer effective therapeutic options for patients with VCP-associated diseases.     

Actomyosin-dependent cortical dynamics contributes to the prophase force-balance in the early Drosophila embryo

Background

The assembly of the Drosophila embryo mitotic spindle during prophase depends upon a balance of outward forces generated by cortical dynein and inward forces generated by kinesin-14 and nuclear elasticity. Myosin II is known to contribute to the dynamics of the cell cortex but how this influences the prophase force-balance is unclear.

Principal Findings

Here we investigated this question by injecting the myosin II inhibitor, Y27632, into early Drosophila embryos. We observed a significant increase in both the area of the dense cortical actin caps and in the spacing of the spindle poles. Tracking of microtubule plus ends marked by EB1-GFP and of actin at the cortex revealed that astral microtubules can interact with all regions of these expanded caps, presumably via their interaction with cortical dynein. In Scrambled mutants displaying abnormally small actin caps but normal prophase spindle length in late prophase, myosin II inhibition produced very short spindles.

Conclusions

These results suggest that two complementary outward forces are exerted on the prophase spindle by the overlying cortex. Specifically, dynein localized on the mechanically firm actin caps and the actomyosin-driven contraction of the deformable soft patches of the actin cortex, cooperate to pull astral microtubules outward. Thus, myosin II controls the size and dynamic properties of the actin-based cortex to influence the spacing of the poles of the underlying spindle during prophase.     

Myocilin Polymorphisms and Primary Open-Angle Glaucoma: A Systematic Review and Meta-Analysis

Background

Glaucoma is the leading cause of irreversible blindness in the world. Recent evidence indicates a role for genetic susceptibility to primary open-angle glaucoma (POAG). The relation between myocilin polymorphisms and POAG susceptibility has been studied in different populations.

Methods

A meta-analysis of 32 published genetic association case-control studies, which examined the relation between POAG and the R46X, R76K, Y347Y, T353I, and Q368X polymorphisms of the myocilin gene, was carried out.

Results

In meta-analysis, significant associations were observed between POAG risk and two myocilin polymorphisms with summarized odds ratio of 4.68 (95%CI, 2.02–10.85) for Q368X and 2.17 (95% CI, 1.32–3.57) for T353I. Both Q368X and T353I were significantly associated with high-tension glaucoma, with summarized odds ratio of 4.26 (1.69, 10.73) and 2.26 (1.37–3.72). In Westerners, significant association was observed for Q368X mutation (odds ratio, 5.17; 95% CI, 2.16–12.40). However, in Asians it was for T353I (odds ratio, 2.17; 95% CI, 1.32–3.57).

Conclusions

There is strong evidence that myocilin polymorphisms are associated with POAG susceptibility, and the prevalence of myocilin mutations might be ethnicity-dependent in Caucasians for Q368X and in Asians for T353I.     

Expression of Abelson interactor 1 (Abi1) correlates with inflammation, KRAS mutation and adenomatous change during colonic carcinogenesis

Background

Abelson interactor 1 (Abi1) is an important regulator of actin dynamics during cytoskeletal reorganization. In this study, our aim was to investigate the expression of Abi1 in colonic mucosa with and without inflammation, colonic polyps, colorectal carcinomas (CRC) and metastases as well as in CRC cell lines with respect to BRAF/KRAS mutation status and to find out whether introduction of KRAS mutation or stimulation with TNFalpha enhances Abi1 protein expression in CRC cells.

Methodology/Principal Findings

We immunohistochemically analyzed Abi1 protein expression in 126 tissue specimens from 95 patients and in 5 colorectal carcinoma cell lines with different mutation status by western immunoblotting. We found that Abi1 expression correlated positively with KRAS, but not BRAF mutation status in the examined tissue samples. Furthermore, Abi1 is overexpressed in inflammatory mucosa, sessile serrated polyps and adenomas, tubular adenomas, invasive CRC and CRC metastasis when compared to healthy mucosa and BRAF-mutated as well as KRAS wild-type hyperplastic polyps. Abi1 expression in carcinoma was independent of microsatellite stability of the tumor. Abi1 protein expression correlated with KRAS mutation in the analyzed CRC cell lines, and upregulation of Abi1 could be induced by TNFalpha treatment as well as transfection of wild-type CRC cells with mutant KRAS. The overexpression of Abi1 could be abolished by treatment with the PI3K-inhibitor Wortmannin after KRAS transfection.

Conclusions/Significance

Our results support a role for Abi1 as a downstream target of inflammatory response and adenomatous change as well as oncogenic KRAS mutation via PI3K, but not BRAF activation. Furthermore, they highlight a possible role for Abi1 as a marker for early KRAS mutation in hyperplastic polyps. Since the protein is a key player in actin dynamics, our data encourages further studies concerning the exact role of Abi1 in actin reorganization upon enhanced KRAS/PI3K signalling during colonic tumorigenesis.     

Regenerative defect in vastus lateralis muscle of patients with chronic obstructive pulmonary disease

Background

Impaired skeletal muscle regeneration could contribute to the progression of muscle atrophy in patients with chronic obstructive pulmonary disease (COPD).

Methods

Satellite cells and myogenesis-related proteins were compared between healthy subjects and patients with COPD, with or without muscle atrophy. Satellite cells were isolated and cultured to assess their proliferative and differentiation aptitudes.

Results

Although satellite cell numbers in muscle samples were similar between groups, the proportion of muscle fibers with central nuclei was increased in COPD. In muscle homogenates, increased expression of MyoD and decreased expression of myogenin and MRF4 were observed in COPD. In cultured satellite cells of patients with COPD, increased protein content was observed for Pax7, Myf5 (proliferation phase) and myogenin (differentiation phase) while myosin heavy chain protein content was significantly lower during differentiation.

Conclusion

In COPD, the number of central nuclei was increased in muscle fibers suggesting a greater number of attempts to regenerate muscle tissue than in healthy subjects. Myogenesis signaling was also altered in muscle homogenates in patients with COPD and there was a profound reduction in the differentiation potential in this population as indicated by a reduced ability to incorporate myosin heavy chain into newly formed myotubes. Collectively, these results indicate that skeletal muscle regenerative capacity termination is impaired in COPD and could contribute to the progression of muscle atrophy progression in this population.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects

Background

Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.

Methodology/Principal Findings

The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.

Conclusions/Significance

Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.