Skeletal Actin Research Articles

Therapeutic Potential of AntagomiR-23b for Treating Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) is a chronically debilitating, rare genetic disease that originates from an expansion of a noncoding CTG repeat in the dystrophia myotonica protein kinase (DMPK) gene. The expansion becomes pathogenic when DMPK transcripts contain 50 or more repetitions due to the sequestration of the muscleblind-like (MBNL) family of proteins. Depletion of MBNLs causes alterations in splicing patterns in transcripts that contribute to clinical symptoms such as myotonia and muscle weakness and wasting. We previously found that microRNA (miR)-23b directly regulates MBNL1 in DM1 myoblasts and mice and that antisense technology (“antagomiRs”) blocking this microRNA (miRNA) boosts MBNL1 protein levels. Here, we show the therapeutic effect over time in response to administration of antagomiR-23b as a treatment in human skeletal actin long repeat (HSALR) mice. Subcutaneous administration of antagomiR-23b upregulated the expression of MBNL1 protein and rescued splicing alterations, grip strength, and myotonia in a dose-dependent manner with long-lasting effects. Additionally, the effects of the treatment on grip strength and myotonia were still slightly notable after 45 days. The pharmacokinetic data obtained provide further evidence that miR-23b could be a valid therapeutic target for DM1.

Adult-Onset Myopathy with Constitutive Activation of Akt following the Loss of hnRNP-U.

SummarySkeletal muscle has the remarkable ability to modulate its mass in response to changes in nutritional input, functional utilization, systemic disease, and age. This is achieved by the coordination of transcriptional and post-transcriptional networks and the signaling cascades balancing anabolic and catabolic processes with energy and nutrient availability. The extent to which alternative splicing regulates these signaling networks is uncertain. Here we investigate the role of the RNA-binding protein hnRNP-U on the expression and splicing of genes and the signaling processes regulating skeletal muscle hypertrophic growth. Muscle-specific Hnrnpu knockout (mKO) mice develop an adult-onset myopathy characterized by the selective atrophy of glycolytic muscle, the constitutive activation of Akt, increases in cellular and metabolic stress gene expression, and changes in the expression and splicing of metabolic and signal transduction genes. These findings link Hnrnpu with the balance between anabolic signaling, cellular and metabolic stress, and physiological growth.

Overexpression of Staufen1 in DM1 mouse skeletal muscle exacerbates dystrophic and atrophic features

In myotonic dystrophy type 1 (DM1), the CUG expansion (CUGexp) in the 3' untranslated region of the dystrophia myotonica protein kinase messenger ribonucleic acid affects the homeostasis of ribonucleic acid-binding proteins, causing the multiple symptoms of DM1. We have previously reported that Staufen1 is increased in skeletal muscles from DM1 mice and patients and that sustained Staufen1 expression in mature mouse muscle causes a progressive myopathy. Here, we hypothesized that the elevated levels of Staufen1 contributes to the myopathic features of the disease. Interestingly, the classic DM1 mouse model human skeletal actin long repeat (HSALR) lacks overt atrophy while expressing CUGexp transcripts and elevated levels of endogenous Staufen1, suggesting a lower sensitivity to atrophic signaling in this model. We report that further overexpression of Staufen1 in the DM1 mouse model HSALR causes a myopathy via inhibition of protein kinase B signaling through an increase in phosphatase tensin homolog, leading to the expression of atrogenes. Interestingly, we also show that Staufen1 regulates the expression of muscleblind-like splicing regulator 1 and CUG-binding protein elav-like family member 1 in wild-type and DM1 skeletal muscle. Together, data obtained from these new DM1 mouse models provide evidence for the role of Staufen1 as an atrophy-associated gene that impacts progressive muscle wasting in DM1. Accordingly, our findings highlight the potential of Staufen1 as a therapeutic target and biomarker.

Cardiac Gene Expression and Histology in a Rat Model of Fat Embolism

IntroductionFat Embolism consequent to Triolein (T) intravenous injection in rats induces severe histopathologic pulmonary damage at 48 hours mostly related to vasculitis, peribronchial and septal inflammation with an increase in mast cells, and eventually, organ fibrosis. We previously reported that the hearts of the same animals at 48 hours did not show vasculitis of coronary arteries branches, inflammation or an increase in mast cells. The objective of this study was to determine if T treatment by 10 weeks induced changes in cardiac gene expression or histopathology related to fat embolism.Materials and Methods24 Sprague Dawley Rats (250–330g) were divided into two groups, 48 hour treatment and 10 week treatment (N=12 each). Six rats of each group were intravenously injected with 0.2 ml of T, and the other 6, 0.2 ml of normal saline. The rats were euthanized under general anesthesia 48 hours or 10 weeks after injection. A cut was made in hearts along the transverse plane; the top half of hearts were snap frozen in liquid nitrogen for gene expression studies and the bottom half was fixed in formaldehyde for histopathological evaluation similar to lung preparations. After RNA isolation, real time RT‐PCR was performed on genes associated with pathological cardiac hypertrophy or fibrosis (beta myosin heavy chain, alpha myosin heavy chain, skeletal muscle alpha actin, atrial natriuretic peptide, brain natriuretic peptide, vimentin, and collagen 1a1).ResultsDespite significant damage to the lungs, there was no significant changes in expression of any of these genes in the hearts of the rats treated with T, versus those of the saline treated group, whether sacrificed at 48 hours or 10 weeks post injection (P>0.05 ANOVA). Histopathological evaluation of the hearts at 10 weeks also did not show any damage in the myocardium in sections stained by H and E and no vasculitis, mast cell increase, or ventricular enlargements. Trichrome staining did not show any fibrotic increase in hearts of T injected animals, thus confirming the previous observations and gene expression analysis.ConclusionThere was a marked contrast in organ damage between hearts and lungs in this study in rats with T treatment. Hearts demonstrated no significant change in gene expression or histology demonstrating a lack of fibroblast infiltration, hypertrophy, or remodeling due to T treatment. Kidney, liver, brains, and aortas also showed varying degrees of histopathological damage compared to the severity of the damage of the lungs. The renin angiotensin system plays a key role in the pathogenesis of the lung damage with fat embolism and this system needs to be further explored for organ differences to fat embolism induced by T treatment.Support or Funding InformationSupported by the Catherine T. Geldmacher Foundation, St. Louis, MO

Tropomyosin isoforms regulate cofilin 1 activity by modulating actin filament conformation

Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F–Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.

Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail.

Myostatin (MSTN) negatively regulates muscle growth and development through inhibiting myoblast proliferation and differentiation. Five alternative splicing isoforms of MSTN (MSTN-A to MSTN-E) have been discovered in domestic avian species. MSTN-A has high expression in skeletal muscle and encodes the full-length peptide with anti-myogenic activity. Another isoform, MSTN-B, is also highly expressed in skeletal muscle and encodes a truncated peptide that has pro-myogenic capabilities in vitro, which include promoting the proliferation and differentiation of quail muscle precursor cells. The objective of this study was to investigate overexpression of MSTN-B in vivo by using two independent lines of transgenic Japanese quail with expression directed in the skeletal muscle. Unexpectedly, the chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior, but not the pectoralis major muscle. Gastrocnemius weight as a percentage of body weight in transgenic quail was increased compared to non-transgenic quail at posthatch day 21 (D21) and posthatch D42. An increase in the size of the gastrocnemius in transgenic quail was attributed to an increase in fiber number but not fiber cross-sectional area (CSA). During embryonic development, paired box 7 (PAX7) expression was prolonged in the transgenic embryos, but other myogenic regulatory factors (MRFs) were unchanged after MSTN-B overexpression. Taken together, these data provide novel insights into the regulation of skeletal muscle development by alternative splicing mechanisms in avians.

Role of the transcription factor Bcl11b in cardiac homeostasis and remodeling

Bcl11b is a zinc finger transcription protein that acts as both a transcriptional repressor and activator in a context dependent manner. A genetic study in humans revealed the association between common genetic variations in the Bcl11b locus and increased risk for cardiovascular disease. Furthermore, microarray data comparisons showed that Bcl11b could modulate the expression of genes during cardiac hypertrophy. To study the role of Bcl11b in the heart under normal and pathological conditions. We conducted a time course analysis on cardiac-specific Bcl11b KO adult mice (Bcl11b-HKO) under normal and hypertrophic conditions (Angiotensin II treatment). Bcl11b-HKO mice showed an acute response marked by the presence of apoptotic cardiomyocytes associated with inflammation, 1 week after Bcl11b inactivation. An increased expression of hypertrophy markers (βMHC, skeletal actin) and pro-apoptotic genes (p53, Bax) was observed in the Bcl11b-HKO mice. Fibrosis-related genes (TGFβ, collagen) were promptly up-regulated, correlating with the formation of fibrotic areas in the Bcl11b-HKO mice over the time. A compromised contractile function, evidenced by a 20% decrease in the ejection fraction, was observed in the Bcl11b-HKO mice at later time points (2 to 6 months after Bcl11b inactivation). Finally, after Angiotensin II treatment, control mice showed a decreased cardiac function. This alteration is mitigated in treated Bcl11b-HKO mice, suggesting a role of Bcl11b in the regulation of cardiac hypertrophy. On-going analysis using isolated adult cardiomyocytes will allow us to decipher the molecular mechanisms affected by Bcl11b inactivation, specifically mechanisms implicated in the apoptosis and hypertrophy. Understanding the role of this protein in cardiac pathology could be clinically relevant and lead to a better understanding of the remodeling process and identify potential targets for pharmacological agents.

Germline or inducible knockout of p300 or CBP in skeletal muscle does not alter insulin sensitivity.

Akt is a critical mediator of insulin-stimulated glucose uptake in skeletal muscle. The acetyltransferases, E1A binding protein p300 (p300) and cAMP response element-binding protein binding protein (CBP) are phosphorylated and activated by Akt, and p300/CBP can acetylate and inactivate Akt, thus giving rise to a possible Akt-p300/CBP axis. Our objective was to determine the importance of p300 and CBP to skeletal muscle insulin sensitivity. We used Cre-LoxP methodology to generate mice with germline [muscle creatine kinase promoter (P-MCK and C-MCK)] or inducible [tamoxifen-activated, human skeletal actin promoter (P-iHSA and C-iHSA)] knockout of p300 or CBP. A subset of P-MCK and C-MCK mice were switched to a calorie-restriction diet (60% of ad libitum intake) or high-fat diet at 10 wk of age. For P-iHSA and C-iHSA mice, knockout was induced at 10 wk of age. At 13-15 wk of age, we measured whole-body energy expenditure, oral glucose tolerance, and/or ex vivo skeletal muscle insulin sensitivity. Although p300 and CBP protein abundance and mRNA expression were reduced 55%-90% in p300 and CBP knockout mice, there were no genotype differences in energy expenditure or fasting glucose and insulin concentrations. Moreover, neither loss of p300 or CBP impacted oral glucose tolerance or skeletal muscle insulin sensitivity, nor did their loss impact alterations in these parameters in response to a calorie restriction or high-fat diet. Muscle-specific loss of either p300 or CBP, be it germline or in adulthood, does not impact energy expenditure, glucose tolerance, or skeletal muscle insulin action.

Myocardial-specific ablation of Jumonji and AT-rich interaction domain–containing 2 (Jarid2) leads to dilated cardiomyopathy in mice

Cardiomyopathy is a common myocardial disease that can lead to sudden death. However, molecular mechanisms underlying cardiomyopathy remain unclear. Jumonji and AT-rich interaction domain-containing 2 (Jarid2) is necessary for embryonic heart development, but functions of Jarid2 after birth remain to be elucidated. Here, we report that myocardial-specific deletion of Jarid2 using αMHC::Cre mice (Jarid2αMHC) causes dilated cardiomyopathy (DCM) and premature death 6-9 months after birth. To determine functions of Jarid2 in the adult heart and DCM, we analyzed gene expression in the heart at postnatal day (p)10 (neonatal) and 7 months (DCM). Pathway analyses revealed that dysregulated genes in Jarid2αMHC hearts at p10, prior to cardiomyopathy, represented heart development and muscle contraction pathways. At 7 months, down-regulated genes in Jarid2αMHC hearts were enriched in metabolic process and ion channel activity pathways and up-regulated genes in extracellular matrix components. In normal hearts, expression levels of contractile genes were increased from p10 to 7 months but were not sufficiently increased in Jarid2αMHC hearts. Moreover, Jarid2 was also necessary to repress fetal contractile genes such as TroponinI1, slow skeletal type (Tnni1) and Actin alpha 2, smooth muscle (Acta2) in neonatal stages through ErbB2-receptor tyrosine kinase 4 (ErbB4) signaling. Interestingly, Ankyrin repeat domain 1 (Ankrd1) and Neuregulin 1 (Nrg1), whose expression levels are known to be increased in the failing heart, were already elevated in Jarid2αMHC hearts within 1 month of birth. Thus, we demonstrate that ablation of Jarid2 in cardiomyocytes results in DCM and suggest that Jarid2 plays important roles in cardiomyocyte maturation during neonatal stages.

Mitochondrial oxidative stress impairs contractile function but paradoxically increases muscle mass via fibre branching.

BackgroundExcess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated.MethodsWe generated mice lacking skeletal muscle‐specific manganese‐superoxide dismutase (mSod2KO) to increase mtROS using a cre‐Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibres and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using in situ and in vitro preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction.ResultsThe mSod2KO mice, contrary to our prediction, exhibit a 10–15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibres (P < 0.05). Despite the increase in muscle mass of gastrocnemius and quadriceps, in situ sciatic nerve‐stimulated isometric maximum‐specific force (N/cm2), force per cross‐sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex‐II‐mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05).ConclusionsIncreased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fibre branching.

Cardiac and skeletal actin substrates uniquely tune cardiac myosin strain-dependent mechanics.

Cardiac ventricular myosin (βmys) translates actin by transducing ATP free energy into mechanical work during muscle contraction. Unitary βmys translation of actin is the step-size. In vitro and in vivo βmys regulates contractile force and velocity autonomously by remixing three different step-sizes with adaptive stepping frequencies. Cardiac and skeletal actin isoforms have a specific 1 : 4 stoichiometry in normal adult human ventriculum. Human adults with inheritable hypertrophic cardiomyopathy (HCM) upregulate skeletal actin in ventriculum probably compensating the diseased muscle's inability to meet demand by adjusting βmys force–velocity characteristics. βmys force–velocity characteristics were compared for skeletal versus cardiac actin substrates using ensemble in vitro motility and single myosin assays. Two competing myosin strain-sensitive mechanisms regulate step-size choices dividing single βmys mechanics into low- and high-force regimes. The actin isoforms alter myosin strain-sensitive regulation such that onset of the high-force regime, where a short step-size is a large or major contributor, is offset to higher loads probably by the unique cardiac essential light chain (ELC) N-terminus/cardiac actin contact at Glu6/Ser358. It modifies βmys force–velocity by stabilizing the ELC N-terminus/cardiac actin association. Uneven onset of the high-force regime for skeletal versus cardiac actin modulates force–velocity characteristics as skeletal/cardiac actin fractional content increases in diseased muscle.

293 - Skeletal muscle specific overexpression of the mitochondrial H2O2 scavenger, peroxiredoxin 3, rescues mitochondrial dysfunction and sarcopenia phenotypes elicited by redox imbalance

Background Reactive oxygen species (ROS) are known to impair contractile function and contribute to muscle atrophy, and one of the primary sources of ROS is mitochondria. The goal of this study is to test whether muscle specific overexpression of mitochondrial hydrogen peroxide scavenger, peroxiredoxin3 (Prdx3), can prevent loss of muscle mass and function associated with sarcopenia. Methods We overexpressed prdx3 by flanking a prdx3 transgene with a stop codon that can be released by skeletal muscle specific expression of cre recombinase driven by the human skeletal actin (HSA) promotor. Muscle specific cre expression allows the transcription of the prdx3 transgene and leads to increased prdx3 expression in skeletal muscle. The prdx3 muscle specific transgenic mice were crossed with an established mouse model of accelerated sarcopenia (Sod1KO) to generate Sod1KO/mPrd3Tg mice. Results In agreement with our previous results, the basal rate of ROS generation was significantly elevated in isolated mitochondria from Sod1KO mice but normalized by prdx3 overexpression in the Sod1KO/mPrd3Tg mice. The reduction in oxygen consumption rate from complex II present in mitochondria from Sod1KO muscle was also restored by prdx3 overexpression. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we tested in vitro contractile properties of extensor digitorum longus (EDL). Maximum isometric specific force was decreased by ~20% in Sod1KO mice, but the reduction in force was rescued inSod1KO/mPrd3Tg mice. Importantly, the loss in gastrocnemius mass that occurs in the Sdo1KO mice was fully restored by prdx3 overexpression. Conclusion Our data demonstrate, for the first time, a direct role of skeletal muscle-specific mitochondrial H2O2 in promoting contractile abnormalities and atrophy of skeletal muscle. This study implicates mitochondrial H2O2 generation as a novel therapeutic target to ameliorate skeletal muscle pathologies.

Muscle specific MnSOD deficiency leads to complex II‐specific inactivity of ETC and contractile dysfunction, but increases muscle mass

Multiple pathological conditions exhibit excess mitochondrial ROS and muscle weakness in parallel, including aging, denervation and cancer cachexia. To directly test the role of mitochondrial ROS in skeletal muscle weakness and atrophy we generated a skeletal muscle specific knockout mouse model in which MnSOD (Sod2), the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre‐Lox approach driven by a constitutively active human skeletal actin promoter (HSA‐Cre). Female wild type and MnSOD deficient (mSod2 KO) mice were studied at 6–8 months of age. In mSod2 KO mice, the observed rate of superoxide generation from isolated skeletal muscle mitochondria was significantly elevated, and hydrogen peroxide generation using permeabilized fibers increased by ~5 fold. ATP generation rate and electron transport chain (ETC) activity were severely reduced in response to Complex II linked substrate, suggesting superoxide‐induced complex II inactivity. Reduced ATP generation rate and ETC activity were consistent with severe exercise intolerance in mSod2 KO mice. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we first tested in situ contractile function using gastrocnemius. Maximum isometric specific force was decreased by ~30 % in mSod2 KO mice with direct stimulation on muscle, but the reduction in force was further diminished (~50%) after stimulation of the sciatic nerve suggesting neuromuscular disruption. Morphological assessment exhibited increases in NMJ size and fragmentation in mSod2 KO mice supporting that mitochondrial dysfunction can feed back to initiate NMJ disruption. Surprisingly, gastrocnemius muscle mass was increased in mSod2 KO mice, which was associated with increased numbers of central nuclei and hyperplasia via fiber branching. In summary, our data suggest that excess mitochondrial superoxide caused a selective loss of complex II activity and mitochondrial defects, leading to severe exercise intolerance. Increased cytoplasmic hydrogen peroxide induced necrosis‐mediated regeneration, which increased muscle mass by fiber branching.Support or Funding InformationNIH/NIA to Dr. Van Remmen (R01 AG050676) and a VA Merit grant to HVRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3β (GSK3β)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3β corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSALR) model]. Here, we show that the inhibition of GSK3β in young HSALR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSALR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3β-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSALR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3β-CUGBP1 pathway in young HSALR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Inhibition of actin polymerization by marine toxin pectenotoxin-2.

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.

Influence of static magnetic fields on human myoblast/mesenchymal stem cell co‑cultures.

The results of surgical repair of extensive muscle tissue defects are still of primary concern, leaving patients with residual cosmetic and functional impairments. Therefore, skeletal muscle tissue engineering attempts to grow functional neo‑tissue from human stem cells to promote tissue regeneration and support defect closure. Despite intensive research efforts, the goal of stable induction of myogenic differentiation in expanded human stem cells by using clinically feasible stimuli, has not yet been reached to a sufficient extent. Therefore, the present study investigated the differentiation potential of static magnetic fields (SMFs), using co‑cultures of human satellite cells and human mesenchymal stem cells (MSCs). It has previously been demonstrated that SMFs may act as a promising myogenic stimulus. Tests were performed on co‑cultures with and without SMF exposure, using growth medium [high growth factor concentrations (GM)] and differentiation medium [low growth factors concentrations (DM)]. AlamarBlue® assay‑based cell proliferation analysis revealed no significant difference between co‑cultures with, vs. without SMF stimulation, regardless of growth factor concentrations in the cell culture medium. To determine the degree of differentiation in co‑cultures under stimulation with SMFs, semi‑quantitative gene expression measurements of the following marker genes were performed: Desmin, myogenic factor 5, myogenic differentiation antigen 1, myogenin, adult myosin heavy chain 1 and skeletal muscle α1 actin. In neither GM nor DM was a steady, significant increase in marker gene expression detected. Verifying the gene expression findings, immunohistochemical antibody staining against differentiation markers revealed that SMF exposure did not enhance myogenic maturation. Therefore, SMF treatment of human satellite cell/MSC co‑cultures did not result in the desired increase in myogenic differentiation. Further studies are required to identify a suitable stimulus for skeletal muscle tissue engineering.

49 - MnSod deficiency causes significant mitochondrial abnormalities and contractile dysfunction in skeletal muscle, but does not decrease muscle mass

Loss of innervation and consequent disruption of the neuromuscular junction (NMJ) in skeletal muscle results in mitochondrial dysfunction and increased ROS that are associated with muscle atrophy and weakness. To directly test the role of mitochondrial dysfunction and ROS in this series of events independent of loss of innervation, we generated a muscle specific knockout mice in which MnSOD, the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre-Lox approach with Cre driven by a constitutively active human skeletal actin promoter (HSA-Cre). We measured mitochondrial ROS production and respiration using permeabilized fibers from red gastrocnemius muscle in wild type and MnSod deficient (mSod2 KO) mice at 6-10 months of age. In mSod2 KO mice, ROS generation was elevated by ~5 fold, and significant defects in mitochondrial respiration were observed-most pronounced in complex II-mediated respiration. Mitochondrial calcium retention capacity was also decreased by 70% in mSod2 KO mice. To determine the effect of reduced MnSOD and increased superoxide on muscle function, we measured in situ contractile function in gastrocnemius muscle. Maximum isometric specific force was decreased by ~30% in mutant mice with direct stimulation on muscle, but the reduction in force was greater (~50%) after stimulation of the sciatic nerve. Surprisingly, gastrocnemius mass was increased in mSod2 KO mice potentially due to compensatory mechanism by fiber branching. Our findings suggest that increased mitochondrial ROS generation and subsequent oxidative stress play an important role in regulating contractile function of skeletal muscle but did not cause muscle atrophy. This result supports the concept that mitochondrial ROS and dysfunction are not critical mediators of denervation-induced atrophy.

Atg5flox-Derived Autophagy-Deficient Model of Pompe Disease: Does It Tell the Whole Story?

Defective autophagy is a prominent feature in Pompe disease, an inherited deficiency of acid alpha-glucosidase. An absence of this enzyme leaves cells unable to digest glycogen in the lysosome. The major tissues affected by glycogen accumulation are cardiac and skeletal muscles. The currently available enzyme replacement therapy (ERT) works well in cardiac, but not in skeletal muscle. The disappointing response to therapy is linked to the presence of large areas of autophagic debris in muscle fibers.

Relaxed and active thin filament structures; a new structural basis for the regulatory mechanism.

The structures of muscle thin filaments reconstituted using skeletal actin and cardiac troponin and tropomyosin have been determined with and without bound Ca2+ using electron microscopy and reference-free single particle analysis. The resulting density maps have been fitted with atomic models of actin, tropomyosin and troponin showing that: (i) the polarity of the troponin complex is consistent with our 2009 findings, with large shape changes in troponin between the two states; (ii) without Ca2+ the tropomyosin pseudo-repeats all lie at almost equivalent positions in the ‘blocked’ position on actin (over subdomains 1 and 2); (iii) in the active state the tropomyosin pseudo-repeats are all displaced towards subdomains 3 and 4 of actin, but the extent of displacement varies within the regulatory unit depending upon the axial location of the pseudo-repeats with respect to troponin. Individual pseudo-repeats with Ca2+ bound to troponin can be assigned either to the ‘closed’ state, a partly activated conformation, or the ‘M-state’, a fully activated conformation which has previously been thought to occur only when myosin heads bind. These results lead to a modified view of the steric blocking model of thin filament regulation in which cooperative activation is governed by troponin-mediated local interactions of the pseudo-repeats of tropomyosin with actin.

OP.2C.01] TETRAHYDROBIOPTERIN REVERSE LEFT VENTRICULAR HYPERTROPHY AND DIASTOLIC DYSFUNCTION THROUGH THE PI3K/P-AKT PATHWAY IN SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Hypertension induced hypertrophy and diastolic dysfunction and is associated with cardiac oxidation and reduced NO production. We hypothesized that tetrahydrobiopterin (BH4) can regulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway and reverse cardiac hypertrophy and diastolic dysfunction in spontaneously hypertensive rats. Design and method: Ten-week-old male spontaneously hypertensive rats (SHR) and age-matched normotensive control Wistar-Kyoto (WKY) rats were divided into five groups, WKY,WKY + BH4, SHR, SHR + BH4 and SHR + VAL. Diastolic function were assessed by echocardiography and hemodynamics. The expression and phosphorylation level of PI3Kand Akt were assayed with western blot analysis. High performance liquid chromatography (HPLC) analysis was used to measure BH4 and biopterins. Quantitative real-time- Polymerase Chain Reaction (PCR) and immunohistochemistry were used to test cardiac hypertrophic genes and expression of Bax and Bcl-2 in the myocardium. Results: In SHR diastolic dysfunction was accompanied by concentric hypertrophy,cardiac oxidation, a reduction in cardiac BH4 and NO production. Administered with BH4 and Valsartan for four weeks, the data showed that both can reverse hypertrophy and improve diastolic function.BH4 and Valsartan blunted the expression of hypertrophy markers alpha - skeletal actin and beta - myosin heavy chain (beta - MHC), only BH4 reduced hypertension induced myocardial fibrosis and expression of transforming growth factor - beta 1 (TGF - beta1). BH 4 reduced cardiac oxidant stress and increased NO production. Exogenous BH 4 increased the phosphorylated Akt level and enhanced expression of Bcl-2. Conclusions: Less BH4 and reduced NO increases myocardial hypertrophy and cardiac oxidative stress, which exacerbates diastolic dysfunction. Exogenous BH4 ameliorates cardiac hypertrophy and diastolic dysfunction through the PI3K/p-Akt pathway. BH4 may be a potent therapy for hypertension with diastolic dysfunction.