Mitochondrial abnormalities contribute to muscle weakness in a Dnajb6 deficient zebrafish model.

Mutations in DNAJB6 are a well-established cause of limb girdle muscular dystrophy type D1 (LGMD D1). Patients with LGMD D1 develop progressive muscle weakness with histology showing fibre damage, autophagic vacuoles, and aggregates. Whilst there are many reports of LGMD D1 patients, the role of DNAJB6 in the muscle is still unclear. In this study, we developed a loss of function zebrafish model in order to investigate the role of Dnajb6. Using a double dnajb6a and dnajb6b mutant model, we show that loss of Dnajb6 leads to a late onset muscle weakness. Interestingly, we find that adult fish lacking Dnajb6 do not have autophagy or myofibril defects, however, they do show mitochondrial changes and damage. This study demonstrates that loss of Dnajb6 causes mitochondrial defects and suggests that this contributes to muscle weakness in LGMD D1. These findings expand our knowledge of the role of Dnajb6 in the muscle and provides a model to screen novel therapies for LGMD D1.

A new role for phosphoinositides in regulating mitochondrial dynamics.

Phosphoinositides are a minor group of membrane-associated phospholipids that are transiently generated on the cytoplasmic leaflet of many organelle membranes and the plasma membrane. There are seven functionally distinct phosphoinositides, each derived via the reversible phosphorylation of phosphatidylinositol in various combinations on the inositol ring. Their generation and termination is tightly regulated by phosphatidylinositol-kinases and -phosphatases. These enzymes can function together in an integrated and coordinated manner, whereby the phosphoinositide product of one enzyme may subsequently serve as a substrate for another to generate a different phosphoinositide species. This regulatory mechanism not only enables the transient generation of phosphoinositides on membranes, but also more complex sequential or bidirectional conversion pathways, and phosphoinositides can also be transferred between organelles via membrane contacts. It is this capacity to fine-tune phosphoinositide signals that makes them ideal regulators of membrane organization and dynamics, through their recruitment of signalling, membrane altering and lipid transfer proteins. Research spanning several decades has provided extensive evidence that phosphoinositides are major gatekeepers of membrane organization, with roles in endocytosis, exocytosis, autophagy, lysosome dynamics, vesicular transport and secretion, cilia, inter-organelle membrane contact, endosome maturation and nuclear function. By contrast, there has been remarkably little known about the role of phosphoinositides at mitochondria - an enigmatic and major knowledge gap, with challenges in reliably detecting phosphoinositides at this site. Here we review recent significant breakthroughs in understanding the role of phosphoinositides in regulating mitochondrial dynamics and metabolic function.

Sequential conversion of PtdIns3P to PtdIns(3,5)P2 via endosome maturation couples nutrient signaling to lysosome reformation and basal autophagy.

Macroautophagy/autophagy occurs basally under nutrient-rich conditions in most mammalian cells, contributing to protein and organelle quality control, and protection against aging and neurodegeneration. During autophagy, lysosomes are heavily utilized via their fusion with autophagosomes and must be repopulated to maintain autophagic degradative capacity. During starvation-induced autophagy, lysosomes are generated via de novo biogenesis under the control of TFEB (transcription factor EB), or by the recycling of autolysosome membranes via autophagic lysosome reformation (ALR). However, these lysosome repopulation processes do not operate under nutrient-rich conditions. In our recent study, we identify a sequential phosphoinositide conversion pathway that enables lysosome repopulation under nutrient-rich conditions to facilitate basal autophagy. Phosphatidylinositol-3,4-bisphosphate (PtdIns[3,4]P2) signals generated downstream of phosphoinositide 3-kinase alpha (PI3Kα) during growth factor stimulation are converted to phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes by INPP4B (inositol polyphosphate-4-phosphatase type II B). We show that PtdIns3P is retained as endosomes mature into endolysosomes, and serves as a substrate for PIKFYVE (phosphoinositide kinase, FYVE-type zinc finger containing) to generate phosphatidylinositol-3,5-bisphosphate (PtdIns[3,5]P2) to promote SNX2-dependent lysosome reformation, basal autophagic flux and protein aggregate degradation. Therefore, endosome maturation couples nutrient signaling to lysosome repopulation during basal autophagy by delivering PI3Kα-derived PtdIns3P to endolysosomes for PtdIns(3,5)P2-dependent lysosome reformation.Abbreviations: ALR: autophagic lysosome reformation; INPP4B: inositol polyphosphate-4-phosphatase type II B; PI3Kα: phosphoinositide 3-kinase alpha; PIKFYVE: phosphoinositide kinase FYVE-type zinc finger containing; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,4)P2: phosphatidylinositol-3,4-bisphosphate; PtdIns(3,5)P2 phosphatidylinositol-3,5-bisphosphate; SNX2 sorting nexin 2; PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3.

Autophagic lysosome reformation in health and disease.

Lysosomes are the primary degradative compartment within cells and there have been significant advances over the past decade toward understanding how lysosome homeostasis is maintained. Lysosome repopulation ensures sustained autophagy function, a fundamental process that protects against disease. During macroautophagy/autophagy, cellular debris is sequestered into phagophores that mature into autophagosomes, which then fuse with lysosomes to generate autolysosomes in which contents are degraded. Autophagy cannot proceed without the sufficient generation of lysosomes, and this can be achieved via their de novo biogenesis. Alternatively, during autophagic lysosome reformation (ALR), lysosomes are generated via the recycling of autolysosome membranes. During this process, autolysosomes undergo significant membrane remodeling and scission to generate membrane fragments, that mature into functional lysosomes. By utilizing membranes already formed during autophagy, this facilitates an efficient pathway for re-deriving lysosomes, particularly under conditions of prolonged autophagic flux. ALR dysfunction is emerging as an important disease mechanism including for neurodegenerative disorders such as hereditary spastic paraplegia and Parkinson disease, neuropathies including Charcot-Marie-Tooth disease, lysosome storage disorders, muscular dystrophy, metabolic syndrome, and inflammatory and liver disorders. Here, we provide a comprehensive review of ALR, including an overview of its dynamic spatiotemporal regulation by MTOR and phosphoinositides, and the role ALR dysfunction plays in many diseases.

Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.

Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.

Skeletal muscle NOX4 is required for adaptive responses that prevent insulin resistance.

Reactive oxygen species (ROS) generated during exercise are considered integral for the health-promoting effects of exercise. However, the precise mechanisms by which exercise and ROS promote metabolic health remain unclear. Here, we demonstrate that skeletal muscle NADPH oxidase 4 (NOX4), which is induced after exercise, facilitates ROS-mediated adaptive responses that promote muscle function, maintain redox balance, and prevent the development of insulin resistance. Conversely, reductions in skeletal muscle NOX4 in aging and obesity contribute to the development of insulin resistance. NOX4 deletion in skeletal muscle compromised exercise capacity and antioxidant defense and promoted oxidative stress and insulin resistance in aging and obesity. The abrogated adaptive mechanisms, oxidative stress, and insulin resistance could be corrected by deleting the H2O2-detoxifying enzyme GPX-1 or by treating mice with an agonist of NFE2L2, the master regulator of antioxidant defense. These findings causally link NOX4-derived ROS in skeletal muscle with adaptive responses that promote muscle function and insulin sensitivity.

Bidirectional interconversion between PtdIns4P and PtdIns(4,5)P2 is required for autophagic lysosome reformation and protection from skeletal muscle disease.

Autophagic lysosome reformation (ALR) recycles autolysosome membranes formed during autophagy, to make lysosomes and is essential for continued autophagy function. Localized membrane remodeling on autolysosomes leads to the extension of reformation tubules, which undergo scission to form new lysosomes. The phosphoinositides phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) induce this remodeling by recruiting protein effectors to membranes. We identified the inositol polyphosphate 5-phosphatase INPP5K, which converts PtdIns(4,5)P2 to PtdIns4P is essential for ALR in skeletal muscle. INPP5K mutations that reduce its 5-phosphatase activity are known to cause muscular dystrophy, via an undefined mechanism. We generated skeletal muscle-specific inpp5k knockout mice which exhibited severe muscle disease, with lysosome depletion and marked autophagy inhibition. This was due to decreased PtdIns4P and increased PtdIns(4,5)P2 on autolysosomes, causing reduced scission of reformation tubules. ALR was restored in cells with loss of INPP5K by expression of wild-type INPP5K, but not muscle-disease causing mutants. Therefore on autolysosomes, both PtdIns(4,5)P2 generation and its removal by INPP5K is required for completion of ALR. Furthermore, skeletal muscle shows a dependence on the membrane recycling ALR pathway to maintain lysosome homeostasis and ensure the protective role of autophagy against disease.

Defective lysosome reformation during autophagy causes skeletal muscle disease.

The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.

Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor Cells.

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.

AKT signaling promotes DNA damage accumulation and proliferation in polycystic kidney disease.

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD, but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3-kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD; however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here, we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD.

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

FHL1 mutants 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.

Identification of FHL1 as a therapeutic target for Duchenne muscular dystrophy.

Utrophin is a potential therapeutic target for the fatal muscle disease, Duchenne muscular dystrophy (DMD). In adult skeletal muscle, utrophin is restricted to the neuromuscular and myotendinous junctions and can compensate for dystrophin loss in mdx mice, a mouse model of DMD, but requires sarcolemmal localization. NFATc1-mediated transcription regulates utrophin expression and the LIM protein, FHL1 which promotes muscle hypertrophy, is a transcriptional activator of NFATc1. By generating mdx/FHL1-transgenic mice, we demonstrate that FHL1 potentiates NFATc1 activation of utrophin to ameliorate the dystrophic pathology. Transgenic FHL1 expression increased sarcolemmal membrane stability, reduced muscle degeneration, decreased inflammation and conferred protection from contraction-induced injury in mdx mice. Significantly, FHL1 expression also reduced progressive muscle degeneration and fibrosis in the diaphragm of aged mdx mice. FHL1 enhanced NFATc1 activation of the utrophin promoter and increased sarcolemmal expression of utrophin in muscles of mdx mice, directing the assembly of a substitute utrophin-glycoprotein complex, and revealing a novel FHL1-NFATc1-utrophin signaling axis that can functionally compensate for dystrophin.

Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer.

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

Evidence for FHL1 as a novel disease gene for isolated hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.

Four and a half LIM protein 1 gene mutations cause four distinct human myopathies: a comprehensive review of the clinical, histological and pathological features.

Mutations in the four and a half LIM protein 1 (FHL1) gene were recently identified as the cause of four distinct skeletal muscle diseases. Since the initial report outlining the first fhl1 mutation in 2008, over 25 different mutations have been identified in patients with reducing body myopathy, X-linked myopathy characterized by postural muscle atrophy, scapuloperoneal myopathy and Emery-Dreifuss muscular dystrophy. Reducing body myopathy was first described four decades ago, its underlying genetic cause was unknown until the discovery of fhl1 mutations. X-linked myopathy characterized by postural muscle atrophy is a novel disease where fhl1 mutations are the only cause. This review will profile each of the FHL1, with a comprehensive analysis of mutations, a comparison of the clinical and histopathological features and will present several hypotheses for the possible disease mechanism(s).

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.

Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy.

Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.

Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy.

Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography-tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle.

Four and a half LIM protein 1: a partner for KCNA5 in human atrium.

AIMS: Protein-protein interactions are critical for the normal membrane trafficking, localization, and function of voltage-gated ion channels. In human heart, the Shaker-related voltage-gated K(+) channel KCNA5 alpha-subunit forms the major basis of an atrial-specific, ultra-rapid delayed rectifier K(+) current, I(Kur). We sought to identify proteins that interact with KCNA5 in human atrium and investigate their role in the I(Kur) complex. METHODS AND RESULTS: Using a glutathione-S-transferase (GST)-KCNA5 C-terminal fusion protein and mass spectrometry-based methods, the scaffolding protein four and a half LIM (for Lin-11, Isl-1, and Mec3) protein 1 (FHL1) was identified as a potential protein partner for KCNA5. Immunoprecipitation experiments confirmed a physical interaction of FHL1 with the K(+) channel complex in human atrium, as well as in Chinese hamster ovary (CHO) cells transfected with both KCNA5 and FHL1. In cotransfected cells, confocal microscopy demonstrated areas of colocalization after immunolabelling both proteins. To investigate the functional effects of this interaction, K(+) currents were recorded in CHO cells transfected with KCNA5 in the absence and presence of FHL1 coexpression. With coexpression of FHL1, K(+) current density was markedly increased, compared with cells expressing KCNA5 alone. This effect was associated with a shift in the voltage dependence of K(+) channel activation to more positive potentials, consistent with findings of I(Kur) in atrial myocytes. FHL1 also increased the extent and speed of K(+) current slow inactivation, with additional effects on the voltage dependence and recovery of this process. CONCLUSION: These results support a role of FHL1 as a key molecular component in the I(Kur) complex in human atrium, where it likely regulates functional expression of KCNA5.