Myosin Heavy Chain Expression Research Articles

PA YEAST SC-1, Polyamine-Rich Saccharomyces cerevisiae, Induces Muscle Hypertrophy in C2C12 Myotubes.

Maintenance of appropriate muscle mass is necessary for good quality of life as skeletal muscles play critical roles in locomotion, metabolic homeostasis, and thermogenesis. Polyamines are essential metabolites that regulate several important cellular functions. In C57BL6 mice who underwent sciatic nerve transection of the hind limb, compensatory muscle hypertrophy is enhanced by the administration of polyamines. However, the action mechanisms of polyamines in muscle hypertrophy remain unclear. Here, we isolated PA YEAST SC-1, a polyamine-rich Saccharomyces cerevisiae, from Baker's yeast. We examined whether PA YEAST SC-1 induces muscle hypertrophy and elucidated the underlying action mechanisms of polyamines and the active ingredients in PA YEAST SC-1 using C2C12 myotubes. PA YEAST SC-1 at 1 mg/mL increased myosin heavy chain expression in C2C12 myotubes. Mechanistically, PA YEAST SC-1 induced the activation of Akt/mechanistic target of rapamycin kinase/p70S6K signaling. Furthermore, PA YEAST SC-1 decreased the expression levels of the ubiquitin ligases, atrogin-1 and muscle RING finger-1, via forkhead box O1 phosphorylation. These findings suggest PA YEAST SC-1 as an effective food ingredient for the treatment of muscle hypertrophy.

Exploring the Potential of Crassostrea nippona Hydrolysates as Dietary Supplements for Mitigating Dexamethasone-Induced Muscle Atrophy in C2C12 Cells.

Muscle atrophy is a detrimental and injurious condition that leads to reduced skeletal muscle mass and disruption of protein metabolism. Oyster (Crassostrea nippona) is a famous and commonly consumed shellfish in East Asia and has become a popular dietary choice worldwide. The current investigation evaluated the efficacy of C. nippona against muscle atrophy, which has become a severe health issue. Mammalian skeletal muscles are primarily responsible for efficient metabolism, energy consumption, and body movements. The proteins that regulate muscle hypertrophy and atrophy are involved in muscle growth. C. nippona extracts were enzymatically hydrolyzed using alcalase (AOH), flavourzyme (FOH), and protamex (POH) to evaluate their efficacy in mitigating dexamethasone-induced muscle damage in C2C12 cells in vitro. AOH exhibited notable cell proliferative abilities, promoting dose-dependent myotube formation. These results were further solidified by protein expression analysis. Western blot and gene expression analysis via RT-qPCR demonstrated that AOH downregulated MuRF-1, Atrogin, Smad 2/3, and Foxo-3a, while upregulating myogenin, MyoD, myosin heavy chain expression, and mTOR, key components of the ubiquitin-proteasome and mTOR signaling pathways. Finally, this study suggests that AOH holds promise for alleviating dexamethasone-induced muscle atrophy in C2C12 cells in vitro, offering insights for developing functional foods targeting conditions akin to sarcopenia.

Spontaneous Alignment of Myotubes Through Myogenic Progenitor Cell Migration.

In large-volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC's natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin-streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We preposition MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

Gromwell ameliorates glucocorticoid-induced muscle atrophy through the regulation of Akt/mTOR pathway

BackgroundMuscle atrophy is characterized by decreased muscle mass, function, and strength. Synthetic glucocorticoids, including dexamethasone (Dexa), are commonly used to treat autoimmune diseases. However, prolonged exposure of Dexa with high dose exerts severe side effects, including muscle atrophy. The purpose of this study was to investigate whether Gromwell root extract (GW) can prevent Dexa-induced muscle atrophy in C2C12 cells and mice and to characterize the composition of GW to identify bioactive compounds.MethodsFor in vitro experiments, GW (0.5 and 1 µg/mL) or lithospermic acid (LA, 5 and 10 µM) was added to C2C12 myotubes on day 4 of differentiation and incubated for 24 h, along with 50 µM Dexa. For in vivo experiment, four-week-old male C57BL/6 mice were randomly divided into the four following groups (n = 7/group): Con group, Dexa group, GW0.1 group, and GW0.2 group. Mice were fed experimental diets of AIN-93 M with or without 0.1 or 0.2% GW for 4 weeks. Subsequently, muscle atrophy was induced by administering an intraperitoneal injection of Dexa at a dose of 15 mg/kg/day for 38 days, in conjunction with dietary intake.ResultsIn Dexa-induced myotube atrophy, treatment with GW increased myotube diameter, reduced the expression of muscle atrophy markers, and enhanced the expression of myosin heavy chain (MHC) isoforms in C2C12 cells. Supplementation with the GW improved muscle function and performance in mice with Dexa-induced muscle atrophy, evidenced in the grip strength and running tests. The GW group showed increased lean body mass, skeletal muscle mass, size, and myosin heavy chain isoform expression, along with reduced skeletal muscle atrophy markers in Dexa-injected mice. Supplementation with GW increased protein synthesis and decreased protein degradation through the Akt/mammalian target of rapamycin and glucocorticoid receptor/forkhead box O3 signaling pathways, respectively. We identified LA as a potential bioactive component of the GW. LA treatment increased myotube diameter and decreased the expression of muscle atrophy markers in Dexa-induced C2C12 cells.ConclusionsThese findings underscore the potential of the GW in preventing Dexa-induced skeletal muscle atrophy and highlight the contribution of LA to its effects.

Characterization of CD90/Thy-1 as a crucial molecular signature for myogenic differentiation in human urine-derived cells through single-cell RNA sequencing

Human urine-derived cells (UDCs) are primary cultured cells originating from the upper urinary tract and are known to be multipotent. We previously developed MYOD1-transduced UDCs (MYOD1-UDCs) as a model recapitulating the pathogenesis of Duchenne muscular dystrophy (DMD) caused by a lack of dystrophin. MYOD1-UDCs also allow evaluation of the efficacy of exon skipping with antisense oligonucleotides. However, despite the introduction of MYOD1, some MYOD1-UDCs failed to form myotubes, possibly because of heterogeneity among UDCs. Here, we carried out single-cell RNA-sequencing analyses and revealed that CD90/Thy-1 was highly expressed in a limited subpopulation of UDCs with high myogenic potency. Furthermore, CD90-positive MYOD1-UDCs, but not CD90-negative cells, could form myotubes expressing high levels of myosin heavy chain and dystrophin. Notably, overexpression of CD90 in CD90-negative MYOD1-UDCs did not enhance myogenic differentiation, whereas CD90 suppression in CD90-positive UDCs led to decreased myotube formation and decreased myosin heavy chain expression. CD90 may thus contribute to the fusion of single-nucleated MYOD1-UDCs into myotubes but is not crucial for promoting the expression of late muscle regulatory factors. Finally, we confirmed that CD90-positive MYOD1-UDCs derived from patients with DMD were a valuable tool for obtaining a highly reproducible and stable evaluation of exon skipping using antisense oligonucleotide.

Dietary short-term supplementation of grape seed proanthocyanidin extract improves pork quality and promotes skeletal muscle fiber type conversion in finishing pigs

Plant extracts are commonly used as feed additives to improve pork quality. However, due to their high cost, shortening the duration of supplement use can help reduce production costs. In this study, we aimed to investigate the effects of grape seed proanthocyanidin extract (GSPE) on meat quality and muscle fiber characteristics of finishing pigs during the late stage of fattening, which was 30 days in our experimental design. The results indicated that short-term dietary supplementation of GSPE significantly reduced backfat thickness, but increased loin eye area and improved meat color and tenderness. Moreover, GSPE increased slow myosin heavy chain (MyHC) expression and malate dehydrogenase (MDH) activity, while decreasing fast MyHC expression and lactate dehydrogenase (LDH) activity in the Longissimus thoracis (LT) muscle. Additionally, GSPE increased the expression of Sirt1 and PGC-1α proteins in the LT muscle of finishing pigs and upregulated AMP-activated protein kinase α 1 (AMPKα1), AMPKα2, nuclear respiratory factor 1 (NRF1), and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) mRNA expression levels. These findings suggest that even during the late stage of fattening, GSPE treatment can regulate skeletal muscle fiber type transformation through the AMPK signaling pathway, thereby affecting the muscle quality of finishing pigs. Therefore, by incorporating GSPE into the diet of pigs during the late stage of fattening, producers can enhance pork quality while reducing production costs.

Abstract C062: Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting

Abstract Background: Patients with pancreatic cancer (PC) often experience cachexia, a multifactorial syndrome that includes weight loss, malnutrition, appetite suppression, and tissue wasting. Unfortunately, the factors secreted from the pancreas or other tissues that drive cachexia are still poorly defined. Lipocalin 2 (LCN2) is a cytokine involved in innate immunity that we have previously shown to be increased in the pancreas and circulation of patients with PC and mouse models of PC. Recent studies suggest that LCN2 contributes to appetite suppression in cancer cachexia in orthotopic models of PC. However, a reduction in muscle regeneration is also commonly seen in cachexia that is not repaired by increased nutrient intake. In a non-cancer setting, LCN2 has been implicated as an important factor in mediating skeletal muscle repair. Therefore, we hypothesized that elevated LCN2 in PC contributes to cachexia by inhibiting muscle progenitor cell proliferation and differentiation into new muscle fibers. Methods: Muscles from a syngeneic orthotopic mouse model of PC were assessed for muscle RING-finger protein-1 (MuRF1, an E3 ubiquitin ligase highly expressed in muscle during wasting) and LCN2 by qPCR. C2C12 mouse muscle myoblasts were cultured in proliferation or differentiation media with recombinant mouse LCN2. Proliferation across 3 days was assessed by MTS assay. Differentiation was assessed by imaging myotubes and western blotting for myosin heavy chain (MyHC) expression which increases during differentiation. PC genetically engineered mice ± a whole body LCN2 were assessed for pancreatic tumor and gastroc muscle size. One-way and Two-way ANOVAs were used to determine statistical significance. Results: LCN2 and MuRF1 expression are elevated in muscles of mice with PC. C2C12 cells treated with LCN2 decreased proliferation in a dose-dependent manner up to concentrations of LCN2 commonly observed in PC. LCN2 treatments at a physiologic concentration observed in PC delayed differentiation of C2C12 cells into myotubes by reducing myotube diameter and delaying MyHC expression. Deleting LCN2 in a PC genetic mouse model prevented body weight and gastroc weight loss but did not affect pancreas weight. Conclusions: Here, we show a relationship between muscle wasting and LCN2 expression during PC. Elevated circulating LCN2 may be contributing to decreased muscle regeneration observed in PC-associated cachexia by directly inhibiting muscle progenitor cell proliferation and differentiation. Targeting LCN2 in PC may be a viable method of slowing or preventing muscle wasting in PC to improve patient outcomes. Citation Format: Kristyn Gumpper-Fedus, Valentina Pita Grisanti, Ericka Velez Bonet, Martha A. Belury, Christopher Coss, Zobeida Cruz-Monserrate. Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C062.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF)

AimsAdiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. Methods and resultsWild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1β, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1β, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. ConclusionThese results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Fluorescent characterization of differentiated myotubes using flow cytometry.

Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM-H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non-intuitive interpretation of laser scatter-based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub-populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, 'rolled-up' myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes 'rolling up' during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub-populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.

Acute Exercise Promptly Normalizes Myocardial Myosin Heavy-Chain Isoform mRNA Composition in Diabetic Rats: Implications for Diabetic Cardiomyopathy.

Background and Objectives: The acute effects of exercise on the myosin heavy-chain (MHC) isoform mRNA expression and the upstream transcription factors in diabetic and non-diabetic hearts remain unexplored. We aimed to determine the acute effect of a single exercise session on the expression of left ventricular MHC, MHC-α and MHC-β, and thyroid receptor (TR), TR-α1 and TR-β, isoform mRNA in diabetic and non-diabetic rats. Materials and Methods: Sprague-Dawley rats were assigned to four groups: non-diabetic control (CS), diabetic exercise (DIEX), sedentary diabetic (DIS), and non-diabetic exercise (CEX). Diabetes was induced via streptozotocin injection (55 mg/kg). DIEX and CEX rats performed an exercise session (60 min at 50 m/min and 0% grade) 6-7 weeks after diabetes induction. Results: MHC-α mRNA was lower in DIS (p = 0.03) and not different in DIEX (p = 0.1) relative to CS. DIS showed higher MHC-β mRNA than the non-diabetic rats, CS and CEX (p = 0.02 and p = 0.009, respectively). MHC-β mRNA in DIEX was normalized to non-diabetic levels in CS (p = 0.3). TR-α1 was higher in DIS and not different in DIEX relative to CS and CEX (p = 0.03 and p = 1.0, respectively). In CEX, exercise did not change MHC-α, MHC-β, and TR-α1 relative to CS (p = 1.0). TR-β was not different between groups. Conclusion: In conclusion, exercise appears to acutely normalize the myocardial MHC and TR isoform mRNA expression only in the diabetic heart. These responses may induce therapeutic mechanisms other than changing the MHC isoform composition.

Combining Porous Se@SiO2 Nanocomposites and dECM Enhances the Myogenic Differentiation of Adipose-Derived Stem Cells.

Volumetric Muscle Loss (VML) denotes the traumatic loss of skeletal muscle, a condition that can result in chronic functional impairment and even disability. While the body can naturally repair injured skeletal muscle within a limited scope, patients experiencing local and severe muscle loss due to VML surpass the compensatory capacity of the muscle itself. Currently, clinical treatments for VML are constrained and demonstrate minimal efficacy. Selenium, a recognized antioxidant, plays a crucial role in regulating cell differentiation, anti-inflammatory responses, and various other physiological functions. We engineered a porous Se@SiO2 nanocomposite (SeNPs) with the purpose of releasing selenium continuously and gradually. This nanocomposite was subsequently combined with a decellularized extracellular matrix (dECM) to explore their collaborative protective and stimulatory effects on the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). The influence of dECM and NPs on the myogenic level, reactive oxygen species (ROS) production, and mitochondrial respiratory chain (MRC) activity of ADSCs was evaluated using Western Blot, ELISA, and Immunofluorescence assay. Our findings demonstrate that the concurrent application of SeNPs and dECM effectively mitigates the apoptosis and intracellular ROS levels in ADSCs. Furthermore, the combination of dECM with SeNPs significantly upregulated the expression of key myogenic markers, including MYOD, MYOG, Desmin, and myosin heavy chain in ADSCs. Notably, this combination also led to an increase in both the number of mitochondria and the respiratory chain activity in ADSCs. The concurrent application of SeNPs and dECM effectively diminishes ROS production, boosts mitochondrial function, and stimulates the myogenic differentiation of ADSCs. This study lays the groundwork for future treatments of VML utilizing the combination of SeNPs and dECM.

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells.

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

Uncoupling Protein 3 Promotes the Myogenic Differentiation of Type IIb Myotubes in C2C12 Cells.

Uncoupling protein 3 (Ucp3) is an important transporter within mitochondria and is mainly expressed in skeletal muscle, brown adipose tissue and the myocardium. However, the effects of Ucp3 on myogenic differentiation are still unclear. This study evaluated the effects of Ucp3 on myogenic differentiation, myofiber type and energy metabolism in C2C12 cells. Gain- and loss-of-function studies revealed that Ucp3 could increase the number of myotubes and promote the myogenic differentiation of C2C12 cells. Furthermore, Ucp3 promoted the expression of the type IIb myofiber marker gene myosin heavy chain 4 (Myh4) and decreased the expression of the type I myofiber marker gene myosin heavy chain 7 (Myh7). In addition, energy metabolism related to the expression of PPARG coactivator 1 alpha (Pgc1-α), ATP synthase, H+ transportation, mitochondrial F1 complex, alpha subunit 1 (Atp5a1), lactate dehydrogenase A (Ldha) and lactate dehydrogenase B (Ldhb) increased with Ucp3 overexpression. Ucp3 could promote the myogenic differentiation of type IIb myotubes and accelerate energy metabolism in C2C12 cells. This study can provide the theoretical basis for understanding the role of Ucp3 in energy metabolism.

Abstract 12750: Three Dimensional Environment for Efficient and Large Scale Generation of Smooth Muscle Cells From HiPSCs

Introduction: Smooth muscle cells (SMCs) play an important role in maintaining vascular homeostasis and the development of diseases. We assess feasibility and efficacy of differentiation human induced pluripotent stem cells (hiPSCs) to SMCs in a 3-dimension (3D). Methods: hiPSCs were cultured in shaker flasks or PBS-0.1 Vessels for 48 h. Differentiation initiated (as day 0) by culturing hiPSC spheroids in RPMI+B27 without insulin (B27-) and CHIR99021 (CHIR) for 24 h. Day 1, medium was replaced with RPMI+B27-, platelet derived growth factor β (PDGF-β), and transforming growth factor β1 (TGFβ1) for 48 h, refreshed medium on day 3 for 48 h. Day 5, differentiating hiPSC-spheroids were dissociated to single cells and cultured as monolayer in RPMI +4% FBS, PDGF-β, and TGFβ1 for 72 h. Refreshed medium day 8 for 72 h. Differentiation efficiency was determined by flow cytometry and fluorescence immunostaining. The biological function of 3D differentiated SMCs was assessed in vitro and in a mouse model of ischemic limb disease in vivo . Results: Both 3D differentiation methods efficiently differentiated hiPSCs into SMCs. Flow cytometry showed that 99.4% cells expressed smooth muscle actin (SMA), 97.9% expressed myosin heavy chain 11 (MYH11), 95.1% expressed SM22, 97.5% expressed calponin, and 97.7% expressed calveolin-3 using shaker flasks. They were 99.6% for SMA, 97.9% for MYH11, 97% for SM22, 89.6% for calponin, and 87.2% for calveolin-3, respectively, using PBS-0.1 Vessels. Mean SMC cell number obtained was 4.56 x 10 7 for shaker flasks and 5.69 x 10 7 for PBS-0.1 Vessels, respectively, which were significantly higher than hiPSCs differentiated in monolayer (5.7x10 6 ). Fluorescence immunostaining confirmed that SMCs differentiated from both methods expressed SMA, MYH11, SM22, Calponin, and calveolin-3. Differentiated SMCs migrated in scratch assay, contracted in response to U-46619, an analog of the endoperoxide prostaglandin, and formed tubular structure on Matrigel in vitro. Fluorescence angiogram imaging showed that SMC transplantation effectively induced perfusion in mouse ischemic limbs. Conclusions: Both 3D differentiation methods, using shaker flasks or PBS-0.1 vessels, efficiently differentiated hiPSCs into large scales of functional SMCs.

Bifidobacterium bifidum and Lactobacillus paracasei alleviate sarcopenia and cognitive impairment in aged mice by regulating gut microbiota-mediated AKT, NF-κB, and FOXO3a signaling pathways

Sarcopenia is closely associated with gut dysbiosis. Probiotics alleviate gut dysbiosis. Therefore, we selected probiotics Lactobacillus paracasei P62 (Lp) and Bifidobacterium bifidum P61 (Bb), which suppressed muscle RING-finger protein-1 (MuRF1) expression and NF-κB activation in C2C12 cells, and examined their effects on muscle mass loss and dysfunction in aged mice. Oral administration of Lp, Bb, or their mix (LB) increased grip strength and treadmill running distance and time. They significantly increased muscle weight in aged mice. They also increased AKT activation, PGC1α, SIRT1, and myosin heavy chain (MyHC) expression, MyHC-positive cell population, and cell size in the gastrocnemius (GA) muscle, while FOXO3a and NF-κB activation, MuRF1, muscle atrophy F-box, and p16 expression, and NF-κB+CD11c+ cell population decreased. Furthermore, they reduced cognitive impairment-like behavior, IL-6 expression, FOXO3a activation, and NF-κB-positive cell population in the hippocampus, GA, and colon, while hippocampal brain-derived neurotropic factor expression increased. They shifted gut microbiota composition in aged mice: they increased Akkermansiaceae and Bacteroidaceae populations, which were positively correlated with total muscle weight and MyHC expression, and decreased Odoribacteraceae and Deferribacteriaceae populations, which were positively correlated with MuRF1 and IL-6 expression. LB alleviated sarcopenia- and cognitive impairment-like symptoms more potently than Lp or Bb alone. Based on these findings, probiotics, particularly Lp, Bb, and LB, can alleviate aging-dependent sarcopenia and cognitive impairment by regulating gut microbiota-mediated AKT, NF-κB, and/or FOXO3a signaling pathways.

Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.

Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We evaluated the effect of RGX365, a protopanaxatriol-type rare ginsenoside mixture, on improving skeletal muscle atrophy. We investigated the myogenic effect of RGX365 on mouse myoblast cells (C2C12) and dexamethasone (10 µM)-induced atrophy of differentiated C2C12. RGX365-treated myotube diameters and myosin heavy chain (MyHC) expression levels were analyzed using immunofluorescence. We evaluated the myogenic effects of RGX365 in aging sarcopenic mice. RGX365 increased myoblast differentiation and MyHC expression, and attenuated the muscle atrophy-inducing F-box (Atrogin-1) and muscle RING finger 1 (MuRF1) expression. Notably, one month of oral administration of RGX365 to 23-month-old sarcopenic mice improved muscle fiber size and the expression of skeletal muscle regeneration-associated molecules. In conclusion, rare ginsenosides, agonists of steroid receptors, can ameliorate skeletal muscle atrophy during long-term administration.

Docosahexaenoic acid (DHA) promotes grass carp (Ctenopharyngodon idella) muscle fiber development by activating MEK/ERK pathway in vitro and in vivo

Docosahexaenoic acid (DHA) is an essential nutrient for aquatic animals and plays a key role in promoting the growth performance of fish. This study was designed to evaluate the effect of DHA on the development of grass carp muscle fiber in vitro and in vivo. In vitro, myoblasts isolated from grass carp were incubated with 50 μM DHA for 24 h. EDU (5-ethynyl-2′ -deoxyuridine) fluorescence staining showed that 50 μM DHA increased the proportion of EDU-positive cells (P < 0.05). And flow cytometry analysis indicated 50 μM DHA also significantly increased the ratio of S and G2 phase cells and decreased the ratio of G1 phase cells (P < 0.05). In addition, expression analysis of proliferation-related genes further showed that 50 μM DHA promoted the proliferation of myoblasts (P < 0.05). Subsequent transcriptome analysis found that the MEK/ERK signaling pathway was activated during this process. Furthermore, the MEK inhibitor trametinib (Tra) inhibited the proliferative effect of DHA on myoblasts, confirming the causal involvement of this pathway. In vivo, 270 fish (initial weight: 27.92 ± 0.02 g) were randomly fed one of the 3 diets for 8 weeks (each diet had 3 replicates): control diet (Con), 1% DHA diet (1% DHA) and the diet supplement with both 1% DHA and 50 mg/kg trametinib (DHA + Tra). The final body weight, specific growth rate, and protein efficiency were significantly increased in the 1% DHA group (P < 0.05). No significant differences were found in crude protein, ash, crude lipid, and moisture levels of the whole body among the 3 groups, whereas the crude protein content in muscle was significantly increased in the 1% DHA group (P < 0.05), suggesting that the increase of FBW was due to muscle growth. Importantly, the fiber density and sarcomere lengths in the 1% DHA group was significantly higher than those of the control and 1% DHA + Tra groups (P < 0.05). The principal component analysis (PCA) on muscle amino acid and fatty acid well separated the DHA group from the control group, and the total weight rank of the muscle amino acid in DHA group appeared to be optimal. The mRNA expression of MEK, ERK and genes related to muscle development significantly increased in the 1% DHA group compared to the control and 1% DHA + Tra groups (P < 0.05). Similarly, the protein expression of myosin heavy chain (MyHc), myoblast determination protein (Myod) and myogenin (Myog) significantly increased in the 1% DHA group. Together, these results suggest that DHA promotes the muscle fiber development likely via the activation of MEK/ERK pathway in grass carp.

Partial Mural Cell Ablation Disrupts Coronary Vasculature Integrity and Induces Systolic Dysfunction.

Background Although the critical role of pericytes in maintaining vascular integrity has been extensively demonstrated in the brain and the retina, little is known about their role in the heart. We aim to investigate structural and functional consequences of partial pericyte depletion (≈60%) in the heart of adult mice. Methods and Results To deplete pericytes in adult mice, we used platelet-derived growth factor receptor β-Cre/ERT2; RosaDTA mice and compared their phenotype with that of control mice (RosaDTA) chosen among their littermates. Cardiac function was assessed via echocardiography and left ventricular catheterization 1 month after the first tamoxifen injection. We found mice depleted with pericytes had a reduced left ventricular ejection fraction and an increased end-diastolic pressure, demonstrating both systolic and diastolic dysfunction. Consistently, mice depleted with pericytes presented a decreased left ventricular contractility and an increased left ventricular relaxation time (dP/dtmin). At the tissue level, mice depleted of pericytes displayed increased coronary endothelium leakage and activation, which was associated with increased CD45+ cell infiltration. Consistent with systolic dysfunction, pericyte depletion was associated with an increased expression of myosin heavy chain 7 and decreased expression of ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 and connexin 43. More important, coculture assays demonstrated, for the first time, that the decreased expression of connexin 43 is likely attributable to a direct effect of pericytes on cardiomyocytes. Besides, this study reveals that cardiac pericytes may undergo strong remodeling on injury. Conclusions Cardiac pericyte depletion induces both systolic and diastolic dysfunction, suggesting that pericyte dysfunction may contribute to the occurrence of cardiac diseases.

Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation.

Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2. C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu. In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles. In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.