On-target action of anti-tropomyosin drugs regulates glucose metabolism.

The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 ß-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2.

We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4Plt53/Plt53 ), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4+/+ ). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking.

Male Presence can Increase Body Mass and Induce a Stress-Response in Female Mice Independent of Costs of Offspring Production.

Sexual reproduction in animals requires close interactions with the opposite sex. These interactions may generate costs of reproduction, because mates can induce detrimental physiological or physical effects on one another, due to their interest in maximising their own fitness. To understand how a male's presence influences aspects of female physiology implicated in reproductive costs in mice, independent of offspring production, we paired females with vasectomised, castrated or intact males, or other females. Being paired with a male, irrespective of his gonadal status, increased female weight. This effect was transient in females paired with castrated males but more persistent in those with vasectomised males. Those paired with males also showed an increase in corticosterone, suggesting an increased stress response. However, this was dependent on the gonadal status of the male housing partner, since those housed with vasectomised males had lower corticosterone than those with castrated males. Altered energy metabolism was only detectable in pregnant females, and oxidative stress was not consistently affected by a female's housing partner. These results suggest that a male's presence alters female weight, and stresses associated with reproduction could be induced by simply the presence of a male, but reduced by mating and/or being solicited to mate.

Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments.

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.

Tropomyosin isoforms support actomyosin biogenesis to generate contractile tension at the epithelial zonula adherens.

Epithelial cells generate contractile forces at their cell-cell contacts. These are concentrated at the specialized apical junction of the zonula adherens (ZA), where a ring of stabilized E-cadherin lies adjacent to prominent actomyosin bundles. Coupling of adhesion and actomyosin contractility yields tension in the junction. The biogenesis of junctional contractility requires actin assembly at the ZA as well as the recruitment of nonmuscle myosin II, but the molecular regulators of these processes are not yet fully understood. We now report a role for tropomyosins 5NM1 (Tm5NM1) and 5NM2 (Tm5NM2) in their generation. Both these tropomyosin isoforms were found at the ZA and their depletion by RNAi or pharmacological inhibition reduced both F-actin and myosin II content at the junction. Photoactivation analysis revealed that the loss of F-actin was attributable to a decrease in filament stability. These changes were accompanied by a decrease in E-cadherin content at junctions. Ultimately, both long-term depletion of Tm5NM1/2 and acute inhibition with drugs caused junctional tension to be reduced. Thus these tropomyosin isoforms are novel contributors to junctional contractility and integrity.

Aged skeletal muscle retains the ability to fully regenerate functional architecture.

While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.

Tropomyosin regulates cell migration during skin wound healing.

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.

Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Diverse roles of the actin cytoskeleton in striated muscle.

In addition to the highly specialized contractile apparatus, it is becoming increasingly clear that there is an extensive actin cytoskeleton which underpins a wide range of functions in striated muscle. Isoforms of cytoskeletal actin and actin-associated proteins (non-muscle myosins, cytoskeletal tropomyosins, and cytoskeletal alpha-actinins) have been detected in a number of regions of striated muscle: the sub-sarcolemmal costamere, the Z-disc and the T-tubule/sarcoplasmic reticulum membranes. As the only known function of these proteins is through association with actin filaments, their presence in striated muscles indicates that there are spatially and functionally distinct cytoskeletal actin filament systems in these tissues. These filaments are likely to have important roles in mechanical support, ion channel function, myofibrillogenenous and vesicle trafficking.

A cytoskeletal tropomyosin can compromise the structural integrity of skeletal muscle.

We have identified a number of extra-sarcomeric actin filaments defined by cytoskeletal tropomyosin (Tm) isoforms. Expression of a cytoskeletal Tm (Tm3) not normally present in skeletal muscle in a transgenic mouse resulted in muscular dystrophy. In the present report we show that muscle pathology in this mouse is late onset (between 2 and 6 months of age) and is predominately in the back and paraspinal muscles. In the Tm3 mice, Evans blue dye uptake in muscle and serum levels of creatine kinase were markedly increased following downhill exercise, and the force drop following a series of lengthening contractions in isolated muscles (extensor digitorum longus) was also significantly increased in these mice. These results demonstrate that expression of an inappropriate Tm in skeletal muscle results in increased susceptibility to contraction-induced damage. The extra-sarcomeric actin cytoskeleton therefore may have an important role in protecting the muscle from contractile stress.

Cytoskeletal tropomyosin Tm5NM1 is required for normal excitation-contraction coupling in skeletal muscle.

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation-contraction coupling in skeletal muscle.

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.

An Actn3 knockout mouse provides mechanistic insights into the association between alpha-actinin-3 deficiency and human athletic performance.

A common nonsense polymorphism (R577X) in the ACTN3 gene results in complete deficiency of the fast skeletal muscle fiber protein alpha-actinin-3 in an estimated one billion humans worldwide. The XX null genotype is under-represented in elite sprint athletes, associated with reduced muscle strength and sprint performance in non-athletes, and is over-represented in endurance athletes, suggesting that alpha-actinin-3 deficiency increases muscle endurance at the cost of power generation. Here we report that muscle from Actn3 knockout mice displays reduced force generation, consistent with results from human association studies. Detailed analysis of knockout mouse muscle reveals reduced fast fiber diameter, increased activity of multiple enzymes in the aerobic metabolic pathway, altered contractile properties, and enhanced recovery from fatigue, suggesting a shift in the properties of fast fibers towards those characteristic of slow fibers. These findings provide the first mechanistic explanation for the reported associations between R577X and human athletic performance and muscle function.

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle.

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.

Tropomyosins in skeletal muscle diseases.

A number of congenital muscle diseases and disorders are caused by mutations in genes that encode the proteins present in or associated with the thin filaments of the muscle sarcomere. These genes include alpha-skeletal actin (ACTA1), beta-tropomyosin (TPM2), alpha-tropomyosin slow (TPM3), nebulin (NEB), troponin I fast (TNNI2), troponin T slow (TNNT1), troponin T fast (TNNT3) and cofilin (CFL2). Mutations in two of the four tropomyosin (Tm) genes, TPM2 and TPM3, result in at least three different skeletal muscle diseases and one disorder as distinguished by the presence of specific clinical features and/or structural abnormalities--nemaline myopathy (TPM2 and TPM3), distal arthrogryposis (TPM2), cap disease (TPM2) and congenital fiber type disproportion (TPM3). These diseases have overlapping clinical features and pathologies and there are cases of family members who have the same mutation, but different diseases (Table 1). The relatively recent discovery of nonmuscle or cytoskeletal Tms in skeletal muscle adds to this complexity since it is now possible that a disease-causing mutation could be in a striated isoform and a cytoskeletal isoform both present in muscle.

Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans.

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein alpha-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associated with elite athlete status and human muscle performance, suggesting that alpha-actinin-3 deficiency influences the function of fast muscle fibers. Here we show that loss of alpha-actinin-3 expression in a knockout mouse model results in a shift in muscle metabolism toward the more efficient aerobic pathway and an increase in intrinsic endurance performance. In addition, we demonstrate that the genomic region surrounding the 577X null allele shows low levels of genetic variation and recombination in individuals of European and East Asian descent, consistent with strong, recent positive selection. We propose that the 577X allele has been positively selected in some human populations owing to its effect on skeletal muscle metabolism.

Parenteral versus enteral nutrition: effect on serum cytokines and the hepatic expression of mRNA of suppressor of cytokine signaling proteins, insulin-like growth factor-1 and the growth hormone receptor in rodent sepsis.

INTRODUCTION: Early nutrition is recommended for patients with sepsis, but data are conflicting regarding the optimum route of delivery. Enteral nutrition (EN), compared with parenteral nutrition (PN), results in poorer achievement of nutritional goals but may be associated with fewer infections. Mechanisms underlying differential effects of the feeding route on patient outcomes are not understood, but probably involve the immune system and the anabolic response to nutrients. We studied the effect of nutrition and the route of delivery of nutrition on cytokine profiles, the growth hormone-insulin-like growth factor-1 (IGF-I) axis and a potential mechanism for immune and anabolic system interaction, the suppressors of cytokine signaling (SOCS), in rodents with and without sepsis. METHODS: Male Sprague-Dawley rats were randomized to laparotomy (Sham) or to cecal ligation and puncture (CLP), with postoperative saline infusion (Starve), with EN or with PN for 72 hours. Serum levels of IL-6 and IL-10 were measured by immunoassay, and hepatic expressions of cytokine-inducible SH2-containing protein, SOCS-2, SOCS-3, IGF-I and the growth hormone receptor (GHR) were measured by real-time quantitative PCR. RESULTS: IL-6 was detectable in all groups, but was only present in all animals receiving CLP-PN. IL-10 was detectable in all but one CLP-PN rat, one CLP-EN rat, approximately 50% of the CLP-Starve rats and no sham-operated rats. Cytokine-inducible SH2-containing protein mRNA was increased in the CLP-EN group compared with the Sham-EN group and the other CLP groups (P < 0.05). SOCS-2 mRNA was decreased in CLP-PN rats compared with Sham-PN rats (P = 0.07). SOCS-3 mRNA was increased with CLP compared with sham operation (P < 0.03). IGF-I mRNA (P < 0.05) and GHR mRNA (P < 0.03) were greater in the fed CLP animals and in the Sham-PN group compared with the starved rats. CONCLUSION: In established sepsis, nutrition and the route of administration of nutrition influences the circulating cytokine patterns and expression of mRNA of SOCS proteins, GHR and IGF-I. The choice of the administration route of nutrition may influence cellular mechanisms that govern the response to hormones and mediators, which further influence the response to nutrients. These findings may be important in the design and analysis of clinical trials of nutritional interventions in sepsis in man.

Does growth hormone allow more efficient nitrogen sparing in postoperative patients requiring parenteral nutrition? A double-blind, placebo-controlled randomised trial.

BACKGROUND & AIMS: Growth hormone (GH) has a strong anabolic effect and is thought to be useful in improving the efficacy of parenteral nutrition (PN) to preserve muscle mass (MM) in the postoperative setting. Unfortunately, the negative clinical outcome of GH treatment in intensive care patients limits its use in this setting, but demands answers to the mechanism behind the action of this therapy. METHOD: In a double-blind randomised controlled study consecutive patients after major abdominal surgery were divided into four groups of either 1/2-PN (0.13 g N/kg/day and 52% of calories as lipid) or full-strength PN (Full-PN) (0.3 g N/kg/day and 65% of calories as lipid) receiving daily injections of either GH (8-16 IU) or placebo for a period of 14 days postoperative. Outcome measures included MM derived from measures of total body potassium (40K counting) and total body nitrogen (TBN) (in vivo neutron capture technique); Fat mass from skin folds; serum insulin like growth factor-I (IGF-I) and its binding proteins (IGFBP). RESULTS: From 43 major upper GI surgical patients randomised 35 completed the study (one patient died from sepsis in the half-strength PN (1/2-PN)+GH group). 1/2-PN (n=11) lost TBN (P=0.001), MM (P=0.005) but not fat. Full-PN (n=9) maintained TBN, MM (P=0.056) and fat. 1/2-PN+GH (n=8) maintained TBN and fat but lost MM (P=0.038). Full-PN+GH (n=7) maintained TBN and MM but lost fat (P=0.018). Two-way ANOVA indicated that PN input (P=0.031) and not GH had a significant effect on MM. GH caused a significant rise in IGF-I levels (290+/-67 and 454+/-71 microg/l for 1/2-PN+GH and Full-PN+GH, respectively) and restored serum IGFBP3 and the acid labile subunit to normal, by the postoperative day 9. CONCLUSION: After major gastrointestinal surgery, GH causes a marked hepatic IGF-I response and nitrogen retention but its effect on body composition was more significant with a high PN input. Further, Full-PN alone was sufficient to prevent nitrogen loss and preserved MM and addition of GH does not provide further metabolic advantage.