A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Effects of 174 G/C polymorphism in the promoter region of the interleukin-6 gene on plasma IL-6 levels and muscle strength in elderly women

We investigated the effect of -174 G/C single-nucleotide polymorphism in the promoter region of the IL6 gene on plasma IL-6 levels and muscle strength, and the relationship between IL-6 levels and muscle strength in elderly women. The sample consisted of 199 elderly residents (73.0 ± 7.8 years old) from rest homes and the community in Belo Horizonte, MG, Brazil. -174 G/C polymorphism was determined by direct sequencing of the product by PCR, and plasma IL-6 concentrations were measured by ELISA. Muscle strength in the knee joint was evaluated using a Biodex System 3 Pro® isokinetic dynamometer. ANCOVA was used to determine the effect of polymorphism on IL-6 levels and muscle strength, and the Pearson correlation coefficient to assess the relationship between IL-6 levels and muscle strength. -174 G/C polymorphism was associated with the plasma IL-6 levels of elderly women (P < 0.01) since homozygotes for the G allele showed high IL-6 levels (GG 3.85 pg/mL, GC + CC 2.13 pg/mL). There was no association of polymorphism on muscle strength (P > 0.05). No association was found between IL-6 levels and knee extensor muscle (r = 0.087, P = 0.306) or flexor (r = -0.011, P = 0.894) strength. An interaction between -174 G/C polymorphism and housing conditions of the sample of elderly women was identified, with the effect of genotype on IL-6 levels being higher in the institutionalized elderly. These results support the evidence that -174 G/C polymorphism of the IL6 gene associates with individual variability of plasma IL-6 levels in elderly women.

The effects of stretching on the flexibility, muscle performance and functionality of institutionalized older women

Stretching has been widely used to increase the range of motion. We assessed the effects of a stretching program on muscle-tendon length, flexibility, torque, and activities of daily living of institutionalized older women. Inclusion/exclusion criteria were according to Mini-Mental State Examination (MMSE) (>13), Barthel Index (>13) and Lysholm Scoring Scale (>84). Seventeen 67 ± 9 year-old elderly women from a nursing home were divided into 2 groups at random: the control group (CG, N = 9) participated in enjoyable cultural activities; the stretching group (SG, N = 8) performed active stretching of hamstrings, 4 bouts of 1 min each. Both groups were supervised three times per week over a period of 8 weeks. Peak torque was assessed by an isokinetic method. Both groups were evaluated by a photogrammetric method to assess muscle-tendon length of uni- and biarticular hip flexors and hamstring flexibility. All measurements were analyzed before and after 8 weeks by two-way ANOVA with the level of significance set at 5%. Hamstring flexibility increased by 30% in the SG group compared to pre-training (76.5 ± 13.0° vs 59.5 ± 9.0°, P = 0.0002) and by 9.2% compared to the CG group (76.5 ± 13.0° vs 64.0 ± 12.0°, P = 0.0018). Muscle-tendon lengths of hip biarticular flexor muscles (124 ± 6.8° vs 118.3 ± 7.6°, 5.0 ± 7.0%, P = 0.031) and eccentric knee extensor peak torque were decreased in the CG group compared to pre-test values (-49.4 ± 16.8 vs -60.5 ± 18.9 Nm, -15.7 ± 20%, P = 0.048). The stretching program was sufficient to increase hamstring flexibility and a lack of stretching can cause reduction of muscle performance.

Plasma cytokine response, lipid peroxidation and NF-κB activation in skeletal muscle following maximum progressive swimming

Our objective was to determine lipid peroxidation and nuclear factor-κB (NF-κB) activation in skeletal muscle and the plasma cytokine profile following maximum progressive swimming. Adult male Swiss mice (N = 15) adapted to the aquatic environment were randomly divided into three groups: immediately after exercise (EX1), 3 h after exercise (EX2) and control. Animals from the exercising groups swam until exhaustion, with an initial workload of 2% of body mass attached to the tail. Control mice did not perform any exercise but were kept immersed in water for 20 min. Maximum swimming led to reactive oxygen species (ROS) generation in skeletal muscle, as indicated by increased thiobarbituric acid reactive species (TBARS) levels (4062.67 ±1487.10 vs 19,072.48 ± 8738.16 nmol malondialdehyde (MDA)/mg protein, control vs EX1). Exercise also promoted NF-κB activation in soleus muscle. Cytokine secretion following exercise was marked by increased plasma interleukin-6 (IL-6) levels 3 h post-exercise (P < 0.05). Interleukin-10 (IL-10) levels were reduced following exercise and remained reduced 3 h post-exercise (P < 0.05). Plasma levels of other cytokines investigated, monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-12 (IL-12), were not altered by exercise. The present findings showed that maximum swimming, as well as other exercise models, led to lipid peroxidation and NF-κB activation in skeletal muscle and increased plasma IL-6 levels. The plasma cytokine response was also marked by reduced IL-10 levels. These results were attributed to exercise type and intensity.

Aerobic exercise training in heart failure: impact on sympathetic hyperactivity and cardiac and skeletal muscle function

Heart failure is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. Chronic neurohumoral excitation (i.e., sympathetic hyperactivity) has been considered to be a hallmark of heart failure and is associated with a poor prognosis, cardiac dysfunction and remodeling, and skeletal myopathy. Aerobic exercise training is efficient in counteracting sympathetic hyperactivity and its toxic effects on cardiac and skeletal muscles. In this review, we describe the effects of aerobic exercise training on sympathetic hyperactivity, skeletal myopathy, as well as cardiac function and remodeling in human and animal heart failure. We also discuss the mechanisms underlying the effects of aerobic exercise training.

Functional and morphological effects of resistance exercise on disuse-induced skeletal muscle atrophy

Abstract The reduction of skeletal muscle loss in pathological states, such as muscle disuse, has considerable effects in terms of rehabilitation and quality of life. Since there is no currently effective and safe treatment available for skeletal muscle atrophy, the search for new alternatives is necessary. Resistance exercise (RE) seems to be an important tool in the treatment of disuse-induced skeletal muscle atrophy by promoting positive functional (strength and power) and structural (hypertrophy and phenotypic changes) adaptive responses. Human and animal studies using different types of resistance exercise (flywheel, vascular occlusion, dynamic, isometric, and eccentric) have obtained results of great importance. However, since RE is a complex phenomenon, lack of strict control of its variables (volume, frequency, intensity, muscle action, rest intervals) limits the interpretation of the impact of the manipulation on skeletal muscle remodeling and function under disuse. The aim of this review is to critically describe the functional and morphological role of resistance exercise in disuse-induced skeletal muscle atrophy with emphasis on the principles of training.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm², pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Longitudinal brain volumetric changes during one year in non-elderly healthy adults: a voxel-based morphometry study

Previous cross-sectional magnetic resonance imaging (MRI) studies of healthy aging in young adults have indicated the presence of significant inverse correlations between age and gray matter volumes, although not homogeneously across all brain regions. However, such cross-sectional studies have important limitations and there is a scarcity of detailed longitudinal MRI studies with repeated measures obtained in the same individuals in order to investigate regional gray matter changes during short periods of time in non-elderly healthy adults. In the present study, 52 healthy young adults aged 18 to 50 years (27 males and 25 females) were followed with repeated MRI acquisitions over approximately 15 months. Gray matter volumes were compared between the two times using voxel-based morphometry, with the prediction that volume changes would be detectable in the frontal lobe, temporal neocortex and hippocampus. Voxel-wise analyses showed significant (P < 0.05, family-wise error corrected) relative volume reductions of gray matter in two small foci located in the right orbitofrontal cortex and left hippocampus. Separate comparisons for males and females showed bilateral gray matter relative reductions in the orbitofrontal cortex over time only in males. We conclude that, in non-elderly healthy adults, subtle gray matter volume alterations are detectable after short periods of time. This underscores the dynamic nature of gray matter changes in the brain during adult life, with regional volume reductions being detectable in brain regions that are relevant to cognitive and emotional processes.

Effects of high-intensity intermittent training on carnitine palmitoyl transferase activity in the gastrocnemius muscle of rats

We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min-1·mg protein-1) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a "time-efficient" strategy inducing metabolic adaptation.

A simple test of muscle coactivation estimation using electromyography

In numerous motor tasks, muscles around a joint act coactively to generate opposite torques. A variety of indexes based on electromyography signals have been presented in the literature to quantify muscle coactivation. However, it is not known how to estimate it reliably using such indexes. The goal of this study was to test the reliability of the estimation of muscle coactivation using electromyography. Isometric coactivation was obtained at various muscle activation levels. For this task, any coactivation measurement/index should present the maximal score (100% of coactivation). Two coactivation indexes were applied. In the first, the antagonistic muscle activity (the lower electromyographic signal between two muscles that generate opposite joint torques) is divided by the mean between the agonistic and antagonistic muscle activations. In the second, the ratio between antagonistic and agonistic muscle activation is calculated. Moreover, we computed these indexes considering different electromyographic amplitude normalization procedures. It was found that the first algorithm, with all signals normalized by their respective maximal voluntary coactivation, generates the index closest to the true value (100%), reaching 92 ± 6%. In contrast, the coactivation index value was 82 ± 12% when the second algorithm was applied and the electromyographic signal was not normalized (P < 0.04). The new finding of the present study is that muscle coactivation is more reliably estimated if the EMG signals are normalized by their respective maximal voluntary contraction obtained during maximal coactivation prior to dividing the antagonistic muscle activity by the mean between the agonistic and antagonistic muscle activations.

Effect of eccentric training on mitochondrial function and oxidative stress in the skeletal muscle of rats

The objective of the present study was to investigate the effects of eccentric training on the activity of mitochondrial respiratory chain enzymes, oxidative stress, muscle damage, and inflammation of skeletal muscle. Eighteen male mice (CF1) weighing 30-35 g were randomly divided into 3 groups (N = 6): untrained, trained eccentric running (16°; TER), and trained running (0°) (TR), and were submitted to an 8-week training program. TER increased muscle oxidative capacity (succinate dehydrogenase and complexes I and II) in a manner similar to TR, and TER did not decrease oxidative damage (xylenol and creatine phosphate) but increased antioxidant enzyme activity (superoxide dismutase and catalase) similar to TR. Muscle damage (creatine kinase) and inflammation (myeloperoxidase) were not reduced by TER. In conclusion, we suggest that TER improves mitochondrial function but does not reduce oxidative stress, muscle damage, or inflammation induced by eccentric contractions.

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1(p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.

Histone demethylase retinoblastoma binding protein 2 regulates the expression of α-smooth muscle actin and vimentin in cirrhotic livers

Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.