Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream (Sparus aurata) Myoblasts.

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as miR-133a/b, miR-206, miR-499, miR-1, and miR-27a. Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

Review: Understanding fish muscle biology in the indeterminate growth species pacu (Piaractus mesopotamicus).

The muscle phenotype of fish is regulated by numerous factors that, although widely explored, still need to be fully understood. In this context, several studies aimed to unravel how internal and external stimuli affect the muscle growth of these vertebrates. The pacu (Piaractus mesopotamicus) is a species of indeterminate muscular growth that quickly reaches high body weight. For this reason, it adds great importance to the productive sector, along with other round fish. In this context, we aimed to compile studies on fish biology and skeletal muscle growth, focusing on studies by our research group that used pacu as an experimental model along with other species. Based on these studies, new muscle phenotype regulators were identified and explored in vivo, in vitro, and in silico studies, which strongly contribute to advances in understanding muscle growth mechanisms with future applications in the productive sector.

Degree of piRNA sharing and Piwi gene expression in the skeletal muscle of Piaractus mesopotamicus (pacu), Colossoma macropomum (tambaqui), and the hybrid tambacu.

PiRNAs are a class of small noncoding RNAs that, in their mature form, bind to Piwi proteins to repress transposable element activity. Besides their role in gametogenesis and genome integrity, recent evidence indicates their action in non-germinative tissues. We performed a global analysis of piRNA and Piwi gene expression in the skeletal muscle of juveniles pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), and the hybrid tambacu to evaluate the degree of piRNA sharing among these three genotypes. Total RNA was sequenced and analyzed using specific parameters of piRNAs by bioinformatics tools. piRNA and Piwi gene expression was analyzed by RT-qPCR. We detected 24 piRNA clusters common to the three genotypes, with eight shared between pacu and tambacu, three between pacu and tambaqui, and five between tambaqui and tambacu; seven, five, and four clusters were unique to pacu, tambacu, and tambaqui, respectively. Genomic localization and fold change values showed two clusters and 100 piRNAs shared among the three genotypes. The gene expression of four piRNAs was evaluated to validate our bioinformatics results. piRNAs from cluster 17 were higher in tambacu than pacu and piRNAs from cluster 18 were more highly expressed in tambacu than tambaqui and pacu. In addition, the expression of Piwis 1 and 2 was higher in tambacu and tambaqui than pacu. Our results open an important window to investigate whether these small noncoding RNAs benefit the hybrid in terms of faster growth and offer a new perspective on the function of piRNAs and Piwis in fish skeletal muscle.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes.

Amino acids (AA) and IGF1 have been demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful for studying muscle regulation, and omics data have contributed enormously to understanding its molecular biology. However, to our knowledge, no study has performed the large-scale sequencing of fish-cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus)-cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. An enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signaling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated, especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs demonstrated a high-level relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates a perspective for improvements in aquaculture and in in vitro meat production.

Transcriptomic Profiling of Rectus Abdominis Muscle in Women with Gestational Diabetes-Induced Myopathy: Characterization of Pathophysiology and Potential Muscle Biomarkers of Pregnancy-Specific Urinary Incontinence.

Gestational diabetes mellitus (GDM) is recognized as a "window of opportunity" for the future prediction of such complications as type 2 diabetes mellitus and pelvic floor muscle disorders, including urinary incontinence and genitourinary dysfunction. Translational studies have reported that pelvic floor muscle disorders are due to a GDM-induced-myopathy (GDiM) of the pelvic floor muscle and rectus abdominis muscle (RAM). We now describe the transcriptome profiling of the RAM obtained by Cesarean section from GDM and non-GDM women with and without pregnancy-specific urinary incontinence (PSUI). We identified 650 genes in total, and the differentially expressed genes were defined by comparing three control groups to the GDM with PSUI group (GDiM). Enrichment analysis showed that GDM with PSUI was associated with decreased gene expression related to muscle structure and muscle protein synthesis, the reduced ability of muscle fibers to ameliorate muscle damage, and the altered the maintenance and generation of energy through glycogenesis. Potential genetic muscle biomarkers were validated by RT-PCR, and their relationship to the pathophysiology of the disease was verified. These findings help elucidate the molecular mechanisms of GDiM and will promote the development of innovative interventions to prevent and treat complications such as post-GDM urinary incontinence.

Integrative microRNAome analysis of skeletal muscle of Colossoma macropomum (tambaqui), Piaractus mesopotamicus (pacu), and the hybrid tambacu, based on next-generation sequencing data.

BACKGROUND: Colossoma macropomum (tambaqui) and Piaractus mesopotamicus (pacu) are good fish species for aquaculture. The tambacu, individuals originating from the induced hybridization of the female tambaqui with the male pacu, present rapid growth and robustness, characteristics which have made the tambacu a good choice for Brazilian fish farms. Here, we used small RNA sequencing to examine global miRNA expression in the genotypes pacu (PC), tambaqui (TQ), and hybrid tambacu (TC), (Juveniles, n = 5 per genotype), to better understand the relationship between tambacu and its parental species, and also to clarify the mechanisms involved in tambacu muscle growth and maintenance based on miRNAs expression. RESULTS: Regarding differentially expressed (DE) miRNAs between the three genotypes, we observed 8 upregulated and 7 downregulated miRNAs considering TC vs. PC; 14 miRNAs were upregulated and 10 were downregulated considering TC vs. TQ, and 15 miRNAs upregulated and 9 were downregulated considering PC vs. TQ. The majority of the miRNAs showed specific regulation for each genotype pair, and no miRNA were shared between the 3 genotype pairs, in both up- and down-regulated miRNAs. Considering only the miRNAs with validated target genes, we observed the miRNAs miR-144-3p, miR-138-5p, miR-206-3p, and miR-499-5p. GO enrichment analysis showed that the main target genes for these miRNAs were grouped in pathways related to oxygen homeostasis, blood vessel modulation, and oxidative metabolism. CONCLUSIONS: Our global miRNA analysis provided interesting DE miRNAs in the skeletal muscle of pacu, tambaqui, and the hybrid tambacu. In addition, in the hybrid tambacu, we identified some miRNAs controlling important molecular muscle markers that could be relevant for the farming maximization.

Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu (Piaractus mesopotamicus) Juveniles: In Vivo and In Vitro Studies.

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu (Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.

Rainbow trout slow myoblast cell culture as a model to study slow skeletal muscle, and the characterization of mir-133 and mir-499 families as a case study.

Muscle fibres are classified as fast, intermediate and slow. In vitro myoblast cell culture model from fast muscle is a very useful tool to study muscle growth and development; however, similar models for slow muscle do not exist. Owing to the compartmentalization of fish muscle fibres, we have developed a slow myoblast cell culture for rainbow trout (Oncorhynchus mykiss). Slow and fast muscle-derived myoblasts have similar morphology, but with differential expression of slow muscle markers such as slow myhc, sox6 and pgc-1α We also characterized the mir-133 and mir-499 microRNA families in trout slow and fast myoblasts as a case study during myogenesis and in response to electrostimulation. Three mir-133 (a-1a, a-1b and a-2) and four mir-499 (aa, ab, ba and bb) paralogues were identified for rainbow trout and named base on their phylogenetic relationship to zebrafish and Atlantic salmon orthologues. Omy-mir-499ab and omy-mir-499bb had 0.6 and 0.5-fold higher expression in slow myoblasts compared with fast myoblasts, whereas mir-133 duplicates had similar levels in both phenotypes and little variation during development. Slow myoblasts also showed increased expression for omy-mir-499b paralogues in response to chronic electrostimulation (7-fold increase for omy-mir-499ba and 2.5-fold increase for omy-mir-499bb). The higher expression of mir-499 paralogues in slow myoblasts suggests a role in phenotype determination, while the lack of significant differences of mir-133 copies during culture development might indicate a different role in fish compared with mammals. We have also found signs of sub-functionalization of mir-499 paralogues after electrostimulation, with omy-mir-499b copies more responsive to electrical signals.

Intense resistance training induces pronounced metabolic stress and impairs hypertrophic response in hind-limb muscles of rats.

Skeletal muscle hypertrophy is an exercise-induced adaptation, particularly in resistance training (RT) programs that use large volumes and low loads. However, evidence regarding the role of rest intervals on metabolic stress and muscular adaptations is inconclusive. Thus, we aimed to investigate the effects of a strenuous RT model (jump-training) on skeletal muscle adaptations and metabolic stress, considering the scarce information about RT models for rats. We hypothesized that jump-training induces metabolic stress and influences negatively the growth of soleus (SOL) and extensor digitorum longus (EDL) muscles of rats. Male Wistar rats (aged 60 days) were randomly assigned to non-trained or trained groups (n = 8/group). Trained rats performed jump-training during 5 days a week for 1, 3, or 5 weeks with 30 s of inter-set rest intervals. Forty-eight hours after the experimental period, rats were euthanized and blood samples immediately drawn to measure creatine kinase activity, lactate and corticosterone concentrations. Muscle weight-to-body weight ratio (MW/BW), cross-sectional area (CSA) and myosin heavy chain (MHC) isoform expression were determined. Higher lactate levels occurred after 20 min of training in weeks 1 and 3. Corticosterone levels were higher after 5 weeks of training. Jump-training had negative effects on hypertrophy of types-I and II muscle fibers after 5 weeks of training, as evidenced by decreased CSA and reduced muscle weight. Our results demonstrated that pronounced metabolic stress and impairment of muscle growth might take place when variables of exercise training are not appropriately manipulated. Lay summary Resistance training (RT) has been used to increase muscle mass. In this regard, training variables (intensity, volume, and frequency) must be strictly controlled in order to evoke substantial muscular fitness. This study shows that rats submitted to 5 weeks of intensive resistance jump-training - high intensity, large volume, and short rest intervals - present high levels of blood corticosterone associated with negative effects on hypertrophy of types-I and II muscle fibers.

The combination of resveratrol and exercise enhances muscle growth characteristics in pacu (Piaractus mesopotamicus).

Pacu is a tropical fish with important value to aquaculture. During cellular metabolism, reactive oxygen species (ROS) are produced, which can influence muscle growth. Resveratrol is an effective antioxidant that scavenges ROS and can modulate physical performance preventing oxidative stress. We investigated the effects of resveratrol and exercise on pacu muscle growth characteristics. Four groups were used: fish fed with control diet /without exercise (C); fish fed with control diet/subjected to exercise (CE); fish fed resveratrol-supplemented diet/without exercise (R); and fish fed resveratrol-supplemented diet/subjected to exercise (RE). At 30 days, the RE group presented a significant increase in body weight, fewer muscle fibers in the 20-40 µm and more fibers in the >60 µm diameter class compared to the C group. At day 7, catalase activity decreased in CE and RE groups. Superoxide dismutase activity decreased only in the CE group. Myod and mtor gene expression was higher in R and RE and igf-1 was up-regulated in the RE group. Murf1a level decreased in CE, R, and RE, while sdha expression was higher in the RE group. We suggest that resveratrol in combination with exercise was beneficial for muscle growth and metabolism, increasing the expression levels of genes related to muscle anabolism and oxidative metabolism, besides the decrease of catabolic gene expression. Notably, all of these changes occurred together with muscle hypertrophy and increased body weight. Our results show a positive application for resveratrol in association with exercise as a strategy to improve the growth performance of juvenile pacus.

The Pathway to Cancer Cachexia: MicroRNA-Regulated Networks in Muscle Wasting Based on Integrative Meta-Analysis.

Cancer cachexia is a multifactorial syndrome that leads to significant weight loss. Cachexia affects 50%-80% of cancer patients, depending on the tumor type, and is associated with 20%-40% of cancer patient deaths. Besides the efforts to identify molecular mechanisms of skeletal muscle atrophy-a key feature in cancer cachexia-no effective therapy for the syndrome is currently available. MicroRNAs are regulators of gene expression, with therapeutic potential in several muscle wasting disorders. We performed a meta-analysis of previously published gene expression data to reveal new potential microRNA-mRNA networks associated with muscle atrophy in cancer cachexia. We retrieved 52 differentially expressed genes in nine studies of muscle tissue from patients and rodent models of cancer cachexia. Next, we predicted microRNAs targeting these differentially expressed genes. We also include global microRNA expression data surveyed in atrophying skeletal muscles from previous studies as background information. We identified deregulated genes involved in the regulation of apoptosis, muscle hypertrophy, catabolism, and acute phase response. We further predicted new microRNA-mRNA interactions, such as miR-27a/Foxo1, miR-27a/Mef2c, miR-27b/Cxcl12, miR-27b/Mef2c, miR-140/Cxcl12, miR-199a/Cav1, and miR-199a/Junb, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting MSTN, CXCL12, and CAMK2B, which may be considered for the development of novel therapeutic strategies for cancer cachexia. Our study has broadened the knowledge of microRNA-regulated networks that are likely associated with muscle atrophy in cancer cachexia, pointing to their involvement as potential targets for novel therapeutic strategies.

Fractal dimension analysis reveals skeletal muscle disorganization in mdx mice.

Duchenne Muscular Dystrophy (DMD) is characterized by muscle extracellular matrix disorganization due to the increased collagen deposition leading to fibrosis that significantly exacerbates disease progression. Fractal dimension analysis is a method that quantifies tissue/cellular disorganization and characterizes complex structures. The first objective of the present study was use fractal analysis to evaluate extracellular matrix disorganization in mdx mice soleus muscle. Next, we mimic a hyper-proliferation of fibrogenic cells by co-culturing NIH3T3 fibroblasts and C2C12 myoblasts to test whether fibroblasts induce disorganization in myoblast arrangement. Here, we show mdx presented high skeletal muscle disorganization as revealed by fractal analysis. Similarly, this method revealed that myoblasts co-cultured with fibroblast also presented cellular arrangement disorganization. We also reanalyzed skeletal muscle microarrays transcriptomic data from mdx and DMD patients that revealed transcripts related to extracellular matrix organization. This analysis also identified Osteoglycin, which was validated as a potential regulator of ECM organization in mdx dystrophic muscles. Our results demonstrate that fractal dimension is useful tool for the analysis of skeletal muscle disorganization in DMD and also reveal a fibroblast-myoblast cross-talk that contributes to "in vitro" myoblast disarrangement.

Influence of postnatal prolactin modulation on the development and maturation of ventral prostate in young rats.

Besides androgenic dependence, other hormones also influence the prostate biology. Prolactin has been described as an important hormone associated with maintenance of prostatic morphophysiology; however, there is a lack of information on the involvement of prolactin during prostate development and growth. This study aimed to evaluate whether perinatal prolactin modulation interferes with rat ventral prostate (VP) development and maturation. Therefore, prolactin or bromocriptine (an inhibitor of prolactin release from the pituitary) were administered to Sprague Dawley rats from postnatal Day (PND) 12 to PND 21 or 35. Animals were then killed and serum hormonal quantification, VP morphological-stereological and immunohistochemical analyses and western blotting reactions were employed. Our results demonstrate that prolactin blockage increased serum testosterone on PND 21, which reflected an increase in anogenital distance. Although prolactin modulation did not interfere with VP weight, it modified VP morphology by dilating the acinar lumen and reducing epithelial cell height. Prolactin activated the signal transducer and activator of transcription (STAT) downstream pathway, increased androgen receptor expression and epithelial proliferation. In addition, prolactin and bromocriptine also increased expression of cytokeratin 18, a marker of luminal-differentiated cells. In conclusion, the VP responds to prolactin modulation through a mechanism of increasing the epithelial proliferative response and dynamics of cell differentiation, especially in animals treated for a more prolonged period.

Low-level laser irradiation induces a transcriptional myotube-like profile in C2C12 myoblasts.

Low-level laser irradiation (LLLI) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLI exerts these effects remain largely unknown. Here, we described global gene expression profiling analysis in C2C12 myoblasts after LLLI that identified 514 differentially expressed genes (DEG). Gene ontology and pathway analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLI with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLI. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLI myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLI myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLI, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLI regulates a set of mRNAs that control myoblast proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLI myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLI on skeletal muscle cells.

Neuromuscular junctions (NMJs): ultrastructural analysis and nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in offspring subjected to protein restriction throughout pregnancy.

Protein restriction during gestation can alter the skeletal muscle phenotype of offspring; however, little is known with regard to whether this also affects the neuromuscular junction (NMJ), as muscle phenotype maintenance depends upon NMJ functional integrity. This study aimed to evaluate the effects of a low protein (6%) intake by dams throughout gestation on male offspring NMJ morphology and nicotinic acetylcholine receptor (nAChR) α1, γ and ε subunit expression in the soleus (SOL) and extensor digitorum longus (EDL) muscles. Four groups of male Wistar offspring rats were studied. The offspring of dams fed low-protein (6% protein, LP) and normal protein (17% protein, NP) diets were evaluated at 30 and 120 days of age, and the SOL and EDL muscles were collected for analysis. Morphological studies using transmission electron microscopy revealed that only SOL NMJs were affected in 30-day-old offspring in the LP group compared with the NP group. SOL NMJs exhibited fewer synaptic folds, the postsynaptic membranes were smooth and myelin figures were also frequently observed in the terminal axons. With regard to the expression of mRNAs encoding nAChR subunits, only 30-day-old LP offspring EDL muscles exhibited reduced α, γ and ε subunit expression compared with the NP group. In conclusion, our results demonstrate that a low-protein diet (6%) imposed throughout pregnancy impairs the expression of mRNAs encoding the nAChR α, γ and ε subunits in EDL NMJs and promotes morphological changes in SOL NMJs of 30-day-old offspring, indicating specific differences among muscle types following long-term maternal protein restriction.

Nitric oxide synthase inhibition impairs muscle regrowth following immobilization.

Nitric oxide (NO) has been shown to increase skeletal muscle protein synthesis. However, the role of NO during skeletal muscle regrowth after immobilization remains unknown. The purpose of this study was to determine whether NO is required for muscle regrowth/recovery after a period of disuse by immobilization. Male Wistar rats were divided into 4 groups: recovered, 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg·kg body mass-1·day-1), NG-nitro-l-arginine methyl ester (l-NAME; 90 mg·kg body mass-1·day-1), and control. The recovered, TRIM, l-NAME groups were submitted to a 7-d muscle recovery period (by remobilization), following a 10-d immobilization period (to induce plantaris [PLA] muscle atrophy). After the experimental period, the PLA muscle was collected for morphometrical (muscle fibers cross-sectional area [CSA]) and molecular (Phospho-mTORSer2448 protein expression) analysis. After 7 d of recovery, the recovered group displayed complete muscle regrowth (CSA, recovered: 2.216 ± 214 vs. CONTROL: 2.219 ± 280 cm2; P > 0.05). However, CSA of the l-NAME (1.911 ± 267 cm2) and TRIM (1.896 ± 219 cm2) groups were statistically (P < 0.05) lower than the recovered and control groups. Additionally, there was a 29% increase in Phos-mTORSer2448 protein expression levels in the recovered group compared to control group, and this increase was blocked in both TRIM and l-NAME groups. In conclusion, our results indicate that NO is crucial for skeletal muscle regrowth after an immobilization period, potentially via the mTOR signaling pathway.

Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus).

BACKGROUND: The Pacu (Piaractus mesopotamicus) is a member of the Characiform family native to the Prata Basin (South America) and a target for the aquaculture industry. A limitation for the development of a selective breeding program for this species is a lack of available genetic information. The primary objectives of the present study were 1) to increase the genetic resources available for the species, 2) to exploit the anatomical separation of myotomal fibres types to compare the transcriptomes of slow and fast muscle phenotypes and 3) to systematically investigate the expression of Ubiquitin Specific Protease (USP) family members in fast and slow muscle in response to fasting and refeeding. RESULTS: We generated 0.6 Tb of pair-end reads from slow and fast skeletal muscle libraries. Over 665 million reads were assembled into 504,065 contigs with an average length of 1,334 bp and N50 = 2,772 bp. We successfully annotated nearly 47% of the transcriptome and identified around 15,000 unique genes and over 8000 complete coding sequences. 319 KEGG metabolic pathways were also annotated and 380 putative microsatellites were identified. 956 and 604 genes were differentially expressed between slow and fast skeletal muscle, respectively. 442 paralogues pairs arising from the teleost-specific whole genome duplication were identified, with the majority showing different expression patterns between fibres types (301 in slow and 245 in fast skeletal muscle). 45 members of the USP family were identified in the transcriptome. Transcript levels were quantified by qPCR in a separate fasting and refeeding experiment. USP genes in fast muscle showed a similar transient increase in expression with fasting as the better characterized E3 ubiquitin ligases. CONCLUSION: We have generated a 53-fold coverage transcriptome for fast and slow myotomal muscle in the pacu (Piaractus mesopotamicus) significantly increasing the genetic resources available for this important aquaculture species. We describe significant differences in gene expression between muscle fibre types for fundamental components of general metabolism, the Pi3k/Akt/mTor network and myogenesis, including detailed analysis of paralogue expression. We also provide a comprehensive description of USP family member expression between muscle fibre types and with changing nutritional status.

Regulation of cardiac microRNAs induced by aerobic exercise training during heart failure.

Exercise training (ET) has beneficial effects on the myocardium in heart failure (HF) patients and in animal models of induced cardiac hypertrophy and failure. We hypothesized that if microRNAs (miRNAs) respond to changes following cardiac stress, then myocardial profiling of these miRNAs may reveal cardio-protective mechanisms of aerobic ET in HF. We used ascending aortic stenosis (AS) inducing HF in Wistar rats. Controls were sham-operated animals. At 18 wk after surgery, rats with cardiac dysfunction were randomized to 10 wk of aerobic ET (HF-ET) or to a heart failure sedentary group (HF-S). ET attenuated cardiac remodeling as well as clinical and pathological signs of HF with maintenance of systolic and diastolic function when compared with that of the HF-S. Global miRNA expression profiling of the cardiac tissue revealed 53 miRNAs exclusively dysregulated in animals in the HF-ET, but only 11 miRNAs were exclusively dysregulated in the HF-S. Out of 23 miRNAs that were differentially regulated in both groups, 17 miRNAs exhibited particularly high increases in expression, including miR-598, miR-429, miR-224, miR-425, and miR-221. From the initial set of deregulated miRNAs, 14 miRNAs with validated targets expressed in cardiac tissue that respond robustly to ET in HF were used to construct miRNA-mRNA regulatory networks that revealed a set of 203 miRNA-target genes involved in programmed cell death, TGF-ß signaling, cellular metabolic processes, cytokine signaling, and cell morphogenesis. Our findings reveal that ET attenuates cardiac abnormalities during HF by regulating cardiac miRNAs with a potential role in cardio-protective mechanisms through multiple effects on gene expression.

Heart failure-induced diaphragm myopathy.

BACKGROUND: Intracellular signaling pathways involved in skeletal myosin heavy chain (MyHC) isoform alterations during heart failure (HF) are not completely understood. We tested the hypothesis that diaphragm expression of mitogen-activated protein kinases (MAPK) and myogenic regulatory factors is changed in rats with myocardial infarction (MI) induced HF. METHODS: Six months after MI rats were subjected to transthoracic echocardiography. After euthanasia, infarcted rats were subdivided in MI/HF- group (with no HF evidence; n=10), and MI/HF+ (with right ventricular hypertrophy and lung congestion; n=10). Sham-operated rats were used as controls (n=10). MyHC isoforms were analyzed by electrophoresis. STATISTICAL ANALYSIS: ANOVA and Pearson correlation. RESULTS: MI/HF- had left cardiac chambers dilation with systolic and diastolic left ventricular dysfunction. Cardiac injury was more intense in MI/HF+ than MI/HF-. MyHC I isoform percentage was higher in MI/HF+ than MI/HF-, and IIb isoform lower in MI/HF+ than Sham. Left atrial diameter-to-body weight ratio positively correlated with MyHC I (p=0.005) and negatively correlated with MyHC IIb (p=0.02). TNF-α serum concentration positively correlated with MyHC I isoform. Total and phosphorylated ERK was lower in MI/HF- and MI/HF+ than Sham. Phosphorylated JNK was lower in MI/HF- than Sham. JNK and p38 did not differ between groups. Expression of NF-κB and the myogenic regulatory factors MyoD, myogenin, and MRF4 was similar between groups. CONCLUSION: Diaphragm MyHC fast-to-slow shift is related to cardiac dysfunction severity and TNF-α serum levels in infarcted rats. Reduced ERK expression seems to participate in MyHC isoform changes. Myogenic regulatory factors and NF-κB do not modulate diaphragm MyHC distribution during chronic HF.