Human Muscle Tissue Research Articles

An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

Histidine-containing dipeptide supplementation improves delayed recall: a systematic review and meta-analysis.

Histidine-containing dipeptides (carnosine, anserine, beta-alanine and others) are found in human muscle tissue and other organs like the brain. Data in rodents and humans indicate that administration of exogenous carnosine improved cognitive performance. However, RCTs results vary. To perform a systematic review and meta-analysis of randomized controlled trials (RCTs) of histidine-containing dipeptide (HCD) supplementation on cognitive performance in humans to assess its utility as a cognitive stabiliser. OVID Medline, Medline, EBM Reviews, Embase, and Cumulative Index to Nursing and Allied Health Literature databases from 1/1/1965 to 1/6/2022 for all RCT of HCDs were searched. 2653 abstracts were screened, identifying 94 full-text articles which were assessed for eligibility. Ten articles reporting the use of HCD supplementation were meta-analysed. The random effects model has been applied using the DerSimonian-Laird method. HCD treatment significantly increased performance on Wechsler Memory Scale (WMS) -2 Delayed recall (Weighted mean difference (WMD) (95% CI (CI)) = 1.5 (0.6, 2.5), P < .01). Treatment with HCDs had no effect on Alzheimer's Disease Assessment Scale-Cognitive (WMD (95% CI) = -0.2 (-1.1, 0.7), P = .65, I2 = 0%), Mini-Mental State Examination (WMD (95% CI) = 0.7 (-0.2, 1.5), P = .14, I2 = 42%), The Wechsler Adult Intelligence Scale (WAIS) Digit span Backward (WMD (95% CI) = 0.1 (-0.3, 0.5), P = .51, I2 = 0%), WAIS digit span Forward (WMD (95% CI) = 0.0 (-0.3, 0.4), P = .85, I2 = 33%) and the WMS-1 Immediate recall (WMD (95% CI) = .7 (-.2, 1.5), P = .11, I2 = 0%). The effect on delayed recall remained in subgroup meta-analysis performed on studies of patients without mild cognitive impairment (MCI), and in those without MCI where average age in the study was above 65. HCD, supplementation improved scores on the Delayed recall examination, a neuropsychological test affected early in Alzheimer's disease. Further studies are needed in people with early cognitive impairment with longer follow-up duration and standardization of carnosine doses to delineate the true effect. PROSPERO registration no. CRD42017075354.

Synthesis, Evaluation of anticancer and antimicrobial activities of some Schiff bases derivatives

In this research, compound [1,2-Bis-(4-amino-5-mercapto-4H-[1,2,4]triazol-3-yl)-ethane-1,2-diol] was used as starting material for synthesis different Schiff's Bases compounds. The fusion of tartaric acid with thiocarbohydrazide produce the compound (C1), the Schiff's bases (C2-C4) were created through the condensation of substances (C1) with different substituted benzaldehydes in presence of glacial acetic acid as catalyst. Thin layer chromatography (TLC) was used to confirm the compounds' purity, and spectroscopic methods were used to infer the compounds' structures (FTIR) and magnetic nuclear resonance spectroscopy (1H-NMR and 13C-NMR). The agar well diffusion method was used to test synthesized compounds for their antibacterial activity against K. pneumonia and S. aureus, and the findings were inconsistent. Target substances were tested for their ability to kill human breast cancer at concentrations of 50 and 100 g/mL. Human muscle tissue HC normal cell line, human cervical cancer Hela cell line, and HePG2 cell line. The results showed that the chemicals had potential cytotoxic activity against the Hela cell line, particularly compound (C4), which had the greatest inhibition at doses of 100 mg/mL among the examined substances&#x0D;

LncRNA LENGA sponges miR-378 to promote myocardial fibrosis in atrial fibrillation.

miR-378 is known to suppress myocardial fibrosis, while its upstream regulators have not been identified. lncRNA LENGA is a recently identified lncRNA in cancer biology. We observed the altered expression of LENGA in atrial fibrillation (AF) patients and predicted its interaction with miR-378. We then explored the interaction between LENGA and miR-378 in AF. Angiotensin-II (Ang-II)-induced human atrial cardiac fibroblasts and human atrial muscle tissues were collected and the expression of LENGA and miR-378 was determined by RT-qPCR. The interaction between LENGA and miR-378 was analyzed through bioinformatics analysis and confirmed by RNA pulldown assay. Cell proliferation and collagen production were analyzed through in vitro assay to analyze the role of LENGA and miR-378 in MF. AF patients showed increased expression of LENGA and deceased expression of miR-378 compared to the sinus rhythm group. LENGA and miR-378 interacted with each other, while they are not closely correlated with each other. Overexpression assay showed that LENGA and miR-378 overexpression failed to affect each other's expression. LENGA promoted collagen production and proliferation of Ang-II-induced atrial fibroblasts, while miR-378 played opposite roles. Moreover, LENGA suppressed the function of miR-378. Therefore, LENGA may sponge miR-378 to promote MF in AF.

Nutrient Rescue of Early Maturing and Deteriorating Satellite Cell-Derived Engineered Muscle Tissue.

Engineered human muscle tissue is a promising tool for tissue models to better understand muscle physiology and diseases, since they can replicate many biomimetic structures and functions of skeletal muscle in vitro. We have developed a method to produce contractile muscle sheet tissues from human myoblasts, based on our cell sheet fabrication technique. This study reports that our tissue engineering technique allowed us to discover unique characteristics of human muscle satellite cells as a cell source for our muscle sheet tissue. The tissues engineered from satellite cells functionally matured within several days, which is earlier than those created from myoblasts. On the other hand, satellite cell-derived muscle sheet tissues were unable to maintain the contractile ability, whereas the myoblast-derived tissues showed muscle contractions for several weeks. The sarcomere structures and membrane-like structures of laminin and dystrophin were lost along with early functional deterioration. Based on a hypothesis that an insufficiency of nutrients caused a shortened lifetime, we supplemented the culture medium for the satellite cell-derived muscle sheet tissues with 10% serum, although a lower serum medium is commonly used to produce muscle tissues. Further combined with the transforming growth factor (TGF-β1) receptor inhibitor, SB431542, the contractile ability of the muscle tissues was increased remarkably and the tissue microstructures were maintained for a longer term, while retaining the early functionalization and the enriched culture conditions prevented early deterioration. These results strengthened our understanding of the biology of myoblasts and satellite cells in muscle tissue formation and provided new insights into the applications of muscle tissue engineering.

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.

Skeletal muscle research is transitioning toward 3D tissue engineered invitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.

Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.

Short-term repeated human biopsy sampling contributes to changes in muscle morphology and higher outcome variability.

Changes in skeletal muscle are an important aspect of overall health. The collection of human muscle to study cellular and molecular processes for research requires a needle biopsy procedure which, in itself, can induce changes in the tissue. To investigate the effect of repeat tissue sampling, we collected skeletal muscle biopsy samples from vastus lateralis separated by 7 days. Cellular infiltrate, central nucleation, enlarged extracellular matrix, and rounding of muscle fibers were used as indices to define muscle damage, and we found that 16/26 samples (61.5%) revealed at least two of these symptoms in the secondary biopsy. The presence of damage influenced outcome measures usually obtained in human biopsies. Damaged muscle showed an increase in the number of small fibers even though average fiber and fiber type-specific cross-sectional area (CSA) were not different. This included higher numbers of embryonic myosin heavy chain-positive fibers (P = 0.001) as well as elevated satellite cell number (P = 0.02) in the damaged areas and higher variability in satellite cell count in the total area (P = 0.04). Collagen content was higher in damaged (P = 0.0003) as well as nondamaged areas (P = 0.05) of the muscle sections of the damaged compared with the nondamaged group. Myofibrillar protein and ribonucleic acid (RNA) fractional synthesis rates were not significantly different between the damaged compared with the nondamaged group. Results indicate that common outcomes as well as outcome variability in human muscle tissue are affected by previous biopsies. Therefore, the extent of potential damage should be assessed when performing repeated biopsies.NEW & NOTEWORTHY Indices of damage can be found in repeated biopsy samples of nonintervened control legs. Variables, directly and not directly related to muscle damage or regeneration, were compromised in second biopsy. There is a need to determine potential damage within muscle tissue when repeated muscle sampling is part of the study design. Muscle biopsy sampling may be a source of increased heterogeneity in human muscle data.

Anaphase-Promoting Complex Subunit 1 Associates with Bone Mineral Density in Human Osteoporotic Bone.

Genome-wide association studies (GWAS) are one of the most common approaches to identify genetic loci that are associated with bone mineral density (BMD). Such novel genetic loci represent new potential targets for the prevention and treatment of fragility fractures. GWAS have identified hundreds of associations with BMD; however, only a few have been functionally evaluated. A locus significantly associated with femoral neck BMD at the genome-wide level is intronic SNP rs17040773 located in the intronic region of the anaphase-promoting complex subunit 1 (ANAPC1) gene (p = 1.5 × 10-9). Here, we functionally evaluate the role of ANAPC1 in bone remodelling by examining the expression of ANAPC1 in human bone and muscle tissues and during the osteogenic differentiation of human primary mesenchymal stem cells (MSCs). The expression of ANAPC1 was significantly decreased 2.3-fold in bone tissues and 6.2-fold in muscle tissue from osteoporotic patients as compared to the osteoarthritic and control tissues. Next, we show that the expression of ANAPC1 changes during the osteogenic differentiation process of human MSCs. Moreover, the silencing of ANAPC1 in human osteosarcoma (HOS) cells reduced RUNX2 expression, suggesting that ANAPC1 affects osteogenic differentiation through RUNX2. Altogether, our results indicate that ANAPC1 plays a role in bone physiology and in the development of osteoporosis.

Unsaturated Long-Chain Fatty Acids Activate Resident Macrophages and Stem Cells in a Human Skeletal Muscle Tissue Model.

Phenotypically heterogeneous populations of tissue-resident macrophages and stem cells play important roles in the regeneration of the skeletal muscle tissue. Previous studies using animal and cell culture models implied a beneficial effect of fatty acid (FA) species on tissue regeneration. Here, we applied a human experimental model using excised muscle tissues from reconstructive surgeries to study the effects of FAs on resident macrophages and stem cells in the natural environment of human skeletal muscle tissue. Muscle tissue samples from 20 donors were included in this study. The expression of 34 cytokines/chemokines was determined, using multiplex protein analysis. The phenotypes of macrophages and stem cells were determined immunohistochemically. The numbers of CD80+ macrophages correlated with the expression levels of IL-1α, IL-1RA, IL-8, IL-17A, and MCP-1, while the PAX7+ and MyoD+ stem cell counts were positively correlated with the expression level of CXCL12α, a recognized chemoattractant for muscle stem cells. Treatment of additional tissue sections with FAs revealed that CD80+ or MARCO+ macrophages- and PAX7+ or MyoD+ stem cells were simultaneously increased by unsaturated long-chain FAs. Taken together, this is the first experimental demonstration of a coordinated activation of macrophages and stem cells in human skeletal muscle tissue.

Fabrication of elliptically constructed liquid crystalline elastomeric scaffolds for 3D artificial tissues

Inspired by the orientation and the fibrous structure of human muscle tissues, we fabricated preconstructed porous liquid crystalline (LC) scaffolds through a two-step polymerization and salt leaching method. A novel strategy combining the aligning properties of LCs and the ease of processing of elastomers for the preparation of elliptical scaffolds for muscle cell culture was proposed in this research. Different from the other types of scaffolds, our biocompatible LC scaffold that can be implanted into the human body using a supporting unit to improve the mechanical properties compared with those of natural muscle. To evaluate the synthesized scaffolds, in vitro experiments using normal human dermal fibroblast (NHDF) cells and smooth muscle cells from rats were carried out, and the sample cells were cultured on each sample scaffold. Based on the results of long-term culture of NHDF cells on the LC scaffolds, it can be confirmed that all three kinds of LC scaffolds have good biocompatibility and provide enough space for cell growth. The addition of gelatin can significantly enhance the biocompatibility of the synthesized scaffolds. Evaluation of scaffold morphologies on cell growth indicates that the molecular arrangement on the scaffolds can induce the growth direction of smooth muscle cells to a certain extent, thereby increasing the formation of highly ordered arrangement tissues. The population doubling time of NHDF cells on the different scaffolds suggest that gelatin can improve the attachment and growth of cells. Investigation of cell viability on LC scaffolds shows that the original LC scaffolds already possess excellent biocompatibility. Additionally, the average cell viability of smooth muscle cells was above 90%, showing that the LC scaffolds in this research are suitable for application in muscle tissue engineering. Based on the results, the gelatin-coated scaffolds are more conducive to the growth of cells in this research and provide promising candidates for tissue engineering in biomedical fields and research fields.

Ascorbic acid reduces Ropivacaine-induced myotoxicity in cultured human osteoporotic skeletal muscle cells

BackgroundOsteoporosis is a worldwide health issue. Loss of bone mass is a potential risk factor for fragility fractures, and osteoporotic fractures place a considerable burden on society. Bone and muscle represent a functional unit in which the two tissues are intimately interconnected. Ropivacaine is a potent local anesthetic used in clinical practice for intraoperative anesthesia and postoperative pain management, in particular for hip surgery. When injected, Ropivacaine can diffuse locally through, in particular in surrounding skeletal muscle tissue, causing dose-dependent cytotoxicity, oxidative stress and myogenesis impairment. Based on those evidences, we focused our attention on Ropivacaine-induced cytotoxicity on cultured human myoblasts.MethodsPrimary human myoblasts and myotubes from healthy subjects, osteoarthritic and osteoporotic patients (OP) were cultured in the presence of Ropivacaine. In some experiments, ascorbic acid (AsA) was added as a potent antioxidant agent. Cell viability and ROS levels were evaluated to investigate the myotoxic activity and Real-Time PCR and Western blot analysis carried out to investigate the expression of proliferation and myogenic markers.ResultsA dose-dependent decrease of cell viability was observed after Ropivacaine exposure in both OP myoblasts and myotubes cultures, whereas those effects were not observed in the presence of Propofol, a general anesthetic. The adding of AsA reduced Ropivacaine negative effects in OP myoblast cultures. In addition, Ropivacaine exposure also increased ROS levels and upregulated Nox4 expression, an enzyme primarily implicated in skeletal muscle ROS generation. AsA treatment counteracted the oxidant activity of Ropivacaine and partially restored the basal condition in cultures. Positive myogenic markers, such as MyoD and Myf5, were downregulated by Ropivacaine exposure, whereas myostatin, a negative regulator of muscle growth and differentiation, was upregulated. The phenotypic deregulation of myogenic controllers in the presence of Ropivacaine was counteracted by AsA treatment.ConclusionsOur findings highlight the oxidative stress-mediated myotoxic effect of Ropivacaine on human skeletal muscle tissue cell cultures, and suggest treatment with AsA as valid strategy to mitigate its negative effects and allowing an ameliorated functional skeletal muscle recovery in patients undergoing hip replacement surgery for osteoporotic bone fracture.

L-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells.

l-Anserine, an imidazole peptide, has a variety of physiological activities, but its effects on skeletal muscle differentiation and muscle contractile force remain unknown. Thus, in this study, we investigated the effect of l-anserine on muscle differentiation and muscle contractile force in human skeletal muscle cells. In two-dimensional culture, 1 μM l-anserine significantly increased the myotube diameters (26.5 ± 1.71, 27.7 ± 1.08, and 28.8 ± 0.85 μm with 0, 0.1, and 1 μM l-anserine, respectively) and the expression levels of genes involved in muscle differentiation and the sarcomere structure. In three-dimensional culture, 1 μM l-anserine significantly increased the contractile force of engineered human skeletal muscle tissues cultured on a microdevice (1.99 ± 0.30, 2.17 ± 0.62, 2.66 ± 0.39, and 3.28 ± 0.85 μN with 0, 0.1, 0.5, and 1 μM l-anserine, respectively). l-Anserine also increased the myotube diameters and the proportion of myotubes with sarcomere structures in the cultured tissues. Furthermore, the histamine receptor 1 (H1R) antagonist attenuated the l-anserine-induced increase in the contractile force, suggesting the involvement of H1R in the mechanism of action of l-anserine. This study showed for the first time that l-anserine enhances muscle differentiation and muscle contractility via H1R.

Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines.

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

Fatty acid amides as potential circulating biomarkers for sarcopenia.

Sarcopenia is characterized by a progressive decrease in skeletal muscle mass and function with age. Given that sarcopenia is associated with various metabolic disorders, effective metabolic biomarkers for its early detection are required. We aimed to investigate the metabolic biomarkers related to sarcopenia in elderly men and perform experimental studies using metabolomics. Plasma metabolites from 142 elderly men, comprising a sarcopenia group and an age-matched control group, were measured using global metabolome profiling. Muscle and plasma samples from an aging mouse model of sarcopenia, as well as cell media and cell lysates during myoblast differentiation, were analysed based on targeted metabolome profiling. Based on these experimental results, fatty acid amides were quantified from human plasma as well as human muscle tissues. The association of fatty acid amide levels with sarcopenia parameters was evaluated. Global metabolome profiling showed that fatty acid amide levels were significantly different in the plasma of elderly men with sarcopenia (all Ps<0.01). Consistent with these results in human plasma, targeted metabolome profiling in an aging mouse model of sarcopenia showed decreased levels of fatty acid amides in plasma but not in muscle tissue. In addition, the levels of fatty acid amides increased in cell lysates during muscle cell differentiation. Targeted metabolome profiling in men showed decreased docosahexaenoic acid ethanolamide (DHA EA) levels in the plasma (P=0.016) but not in the muscle of men with sarcopenia. DHA EA level was positively correlated with sarcopenia parameters such as skeletal muscle mass index (SMI) and handgrip strength (HGS) (P=0.001, P=0.001, respectively). The area under the receiver-operating characteristic curve (AUC) for DHA EA level≤4.60 fmol/μL for sarcopenia was 0.618 (95% confidence interval [CI]: 0.532-0.698). DHA EA level≤4.60 fmol/μL was associated with a significantly greater likelihood of sarcopenia (odds ratio [OR]: 2.11, 95% CI: 1.03-4.30), independent of HGS. The addition of DHA EA level to age and HGS significantly improved the AUC from 0.620 to 0.691 (P=0.0497). Our study demonstrated that fatty acid amides are potential circulating biomarkers in elderly men with sarcopenia. DHA EA, in particular, strongly related to muscle mass and strength, can be a key metabolite to become a reliable metabolic biomarker for sarcopenia. Further research on fatty acid amides will provide insights into the metabolomic changes relevant to sarcopenia from an aging perspective.

MicroRNA expression profile analysis in human skeletal muscle tissue: Selection of critical reference

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs, that modulate gene expression by targeting messenger RNA of many processes. Thus, miRNAs are key regulators of both physiological and pathological settings. Reliable results of quantitative miRNA evaluation depend on suitable reference genes (RGs) for data normalization. To date, no consensus has been reached on the best RG for muscle tissue. We assessed RGs stability in skeletal muscle tissue in patients with spinal deformity. Ninety tissue samples were obtained from the deep paravertebral muscles from the convex and concave sides of the spinal curvature, as well as the superficial paraspinal muscles. We evaluated the stability of twelve miRNAs (hsa-miR-1–3p, hsa-miR-1–5p, hsa-miR-26b-5p, hsa-miR-92a-3p, hsa-miR-133a-3p, hsa-miR-133a-5p, hsa-miR-133b, hsa-miR-191–5p, hsa-miR-206, hsa-miR-208b-5p, hsa-miR-486–5p, hsa-miR-499a-5p), finding three to be indicative of reference miRNA, and nine as muscle-tissue specific. Stability was quantified using four statistical tools and a comprehensive ranking system. Three miRNAs were indicated as the most stable, and we assessed hsa-miR-486–5p as the most, and hsa-miR-208b-5p as the least suitable RGs for miRNA quantitative analyses. We recommend using a minimum of three RGs miRNA to normalize RT-qPCR data. Finally, qPCR efficiency should always be considered. To obtain consistent results, data normalization in muscle tissue is required.

High-quality nuclei isolation from postmortem human heart muscle tissues for single-cell studies

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay

Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novel Arp5 mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonical Arp5 isoform, i.e., Arp5(7a), to the NMD-targeted isoform Arp5(7b) occurred during differentiation, suggesting that Arp5 expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of both Arp5 isoforms and measured higher levels of Arp5(7b) in muscle and brain tissues, where ARP5 is less expressed. The 3′ splice site in Arp5 exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, the Arp5(7b) isoform was barely detectable. The expression of several splicing factors involved in 3′ splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels of Arp5(7b) and decreased the expression of Arp5(7a). Furthermore, strong positive correlations were found between Arp5 expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus, Arp5 expression in muscle tissues is most likely regulated by the AS-NMD pathway.

Intra- and inter-session reliability and repeatability of an infrared thermography device designed for materials to measure skin temperature of the triceps surae muscle tissue of athletes.

Infrared thermography devices have been commonly applied to measure superficial temperature in structural composites and walls. These tools were cheaper than other thermographic devices used to measure superficial human muscle tissue temperature. In addition, infrared thermography has been previously used to assess skin temperature related to muscle tissue conditions in the triceps surae of athletes. Nevertheless, the reliability and repeatability of an infrared thermography device designed for materials, such as the Manual Infrared Camera PCE-TC 30, have yet to be determined to measure skin temperature of the triceps surae muscle tissue of athletes. The purpose was to determine the procedure's intra- and inter-session reliability and repeatability to determine skin temperature within the Manual Infrared Camera PCE-TC 30 thermography device in the triceps surae muscle tissue of athletes, which was initially designed to measure the superficial temperature of materials. A total of 34 triceps surae muscles were bilaterally assessed from 17 healthy athletes using the Manual Infrared Camera PCE-TC 30 thermography device to determine intra- (at the same day separated by 1 h) and inter-session (at alternate days separated by 48 h) reliability and repeatability of the skin temperature of the soleus, medial and lateral gastrocnemius muscles. The triceps surae complex weas measured by a region of interest of 1 cm2 through five infrared thermography images for each muscle. Statistical analyses comprised intraclass correlation coefficient (ICC), standard error of measurement (SEM), minimum detectable change (MCD), systematic error of measurement, correlation (r), and Bland-Altman plots completed with linear regression models (R 2). Intra- and inter-session measurements of the proposed infrared thermography procedure showed excellent reliability (ICC(1,2) = 0.968-0.977), measurement errors (SEM = 0.186-0.232 °C; MDC = 0.515-0.643 °C), correlations (r = 0.885-0.953), and did not present significant systematic error of measurements (P > 0.05). Adequate agreement between each pair of measurement moments was presented by the Bland-Altman plots according to the limits of agreement and non-significant linear regression models (R 2 = 0.000-0.019; P > 0.05). The proposed procedure to determine skin temperature within the Manual Infrared Camera PCE-TC 30 thermography device presented excellent intra- and inter-session reliability and repeatability in athletes' triceps surae muscle tissue. Future studies should consider the SEM and MDC of this procedure to measure the skin temperature of soleus, medial, and lateral gastrocnemius muscles to promote triceps surae muscle prevention and recovery in athletes.

The protective effect of cannabinoids against colorectal cancer cachexia through modulation of inflammation and immune responses

Cancer cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, and there are currently no FDA-approved medications. In the present study, upregulation of six cytokines was observed in serum samples from patients with colorectal cancer (CRC) and in mouse models. A negative correlation between the levels of the six cytokines and body mass index in CRC patients was seen. Gene Ontology analysis revealed that these cytokines were involved in regulating T cell proliferation. The infiltration of CD8+ T cells was found to be associated with muscle atrophy in mice with CRC. Adoptive transfer of CD8+ T cells isolated from CRC mice resulted in muscle wasting in recipients. The Genotype-Tissue Expression database showed that negative correlations between the expression of cachexia markers and cannabinoid receptor 2 (CB2) in human skeletal muscle tissues. Pharmacological treatment with Δ9-tetrahydrocannabinol (Δ9-THC), a selective CB2 agonist or overexpression of CB2 attenuated CRC-associated muscle atrophy. In contrast, knockout of CB2 with a CRISPR/Cas9-based strategy or depletion of CD8+ T cells in CRC mice abolished the Δ9-THC-mediated effects. This study demonstrates that cannabinoids ameliorate CD8+ T cell infiltration in CRC-associated skeletal muscle atrophy via a CB2-mediated pathway. Serum levels of the six-cytokine signature might serve as a potential biomarker to detect the therapeutic effects of cannabinoids in CRC-associated cachexia.