Myocyte Differentiation Research Articles

OR-050 The Development of a Protein Powder Product with an integrated solution for Active Population

Objective Sports nutrition is expanding from professional athletes to a broader spectrum of consumers focused on fitness and improving overall wellness. The recommendation of combining exercise and healthy diet has been highlighted by Health China 2030 and National Nutrition Planning, leading to increased embrace of active lifestyle among Chinese people. Along with the increasing involvement in sports comes with concerns on decreasing resting metabolic rate due to weight loss, impaired joint health and post-exercise immunological response. To confront the above-mentioned challenges, we aimed to develop a product with an integrated solution to support fitness goals while diminishing exercise-related concerns.
 Methods Literature review was conducted for core ingredient screen, followed by in vitro studies to substantiate and compare efficacy of ingredients from various resources. Muscle protein synthesis was measured in myotubes during the differentiation and fusion of C2C12 myocytes. Myotube fusion density was quantified by ImageJ, an image processing program, subsequent to myotube staining.
 Results Muscle mass gain enhances resting metabolic rate. It has been shown that whey protein supplementation at 20 g/d significantly increased lean muscle synthesis rate in post-exercise subjects. The efficacy of whey protein powder sourced from different resources were compared by conducting in vitro studies. A large body of evidence has shown that collagen peptide supplementation can mitigate joint pain and maintain joint health in athletic population. Yeast β-glucan was spotlighted as an immunity enhancer with antimicrobial protection and benefit in minimizing post-exercise immunosuppression. 
 Conclusions A protein powder containing over 20 g/serving whey protein, combining with collagen peptide and yeast β-glucan may convey beneficial effects in muscle synthesis, as well as protective effects against joint pain and post-exercise immunosuppression among active population. The combination of the above-mentioned ingredients could provide a well-round solution to confront the challenges that met by most active population.

Mir-206 partially rescues myogenesis deficiency by inhibiting CUGBP1 accumulation in the cell models of myotonic dystrophy

ABSTRACTObjectives: In this study, we aim to determine how CUG-expansion and the abundance of Celf1 regulates normal myocyte differentiation and reveal the role ofmiR-206 in myotonic dystrohy and explore a possible gene therapy vector. Methods: we set up CUG-expansion and Celf1 overexpression C2C12 cell models to imitate the myocyte differentiation defects of DM1, then transfected AdvmiR-206 into cell models, tested the level of myogenic markers MyoD, MyoG, Mef2c, Celf1 by RT-PCRand Western Blotting, detected myotube formation by myosin heavy chain immunostaining. Result: 3′-UTR CUG-expansion leads to myotube defects and impaired myoblasts differentiation. Overexpression of Celf1 inhibits myoblast differentiation and impairs differentiation. Knockdown of Celf1 partially rescues differentiation defects of myoblasts harboring CUG-expansion. miR-206 incompletely reverses myoblast differentiation inhibition induced by CUG-expansion and partially recuses myoblast differentiation defects induced by Celf1 overexpression. Conclusions: Ectopic miR-206 mimicking the endogenous temporal patterns specifically drives a myocyte program that boosts myoblast lineages, likely by promoting the expression of MyoD to rectify the myogenic deficiency by stimulating the accumulation of Celf1.Abbreviations: DMPK: (dystrophia myotonica protein kinase); 3′-UTR: (3′-untranslated region); MBNL1: (muscleblind-like [Drosophila]); DM1: (myotonic dystrophy type 1); GFP: (green fluorescent protein); RT-PCR: (quantitative reverse transcriptase-polymerase chain reaction); shRNA: (short hairpin RNA)

New Insights Into the Long Non-coding RNA SRA: Physiological Functions and Mechanisms of Action.

Long non-coding RNAs (lncRNA) are emerging as new genetic/epigenetic regulators that can impact almost all physiological functions. Here, we focus on the long non-coding steroid receptor RNA activator (SRA), including new insights into its effects on gene expression, the cell cycle, and differentiation; how these relate to physiology and disease; and the mechanisms underlying these effects. We discuss how SRA acts as an RNA coactivator in nuclear receptor signaling; its effects on steroidogenesis, adipogenesis, and myocyte differentiation; the impact on breast and prostate cancer tumorigenesis; and, finally, its ability to modulate hepatic steatosis through several signaling pathways. Genome-wide analysis reveals that SRA regulates hundreds of target genes in adipocytes and breast cancer cells and binds to thousands of genomic sites in human pluripotent stem cells. Recent studies indicate that SRA acts as a molecular scaffold and forms networks with numerous coregulators and chromatin-modifying regulators in both activating and repressive complexes. We discuss how modifications to SRA's unique stem-loop secondary structure are important for SRA function, and highlight the various SRA isoforms and mutations that have clinical implications. Finally, we discuss the future directions for better understanding the molecular mechanisms of SRA action and how this might lead to new diagnostic and therapeutic approaches.

Abstract 428: Cytoglobin: A Novel Regulator of the DNA Damage Response Within Cardiomyocytes

Rationale: Oxidative stress is a major determinant of DNA damage within cardiomyocytes both at baseline and following cardiac injury. Cytoglobin (Cygb), a stress responsive hemoprotein, serves as a redox modulator and imparts cytoprotection in a variety of cell types. In addition, Cygb is involved in regulation of myocyte proliferation and differentiation due to its ability to scavenge free radicals and maintain redox equilibrium within the myocytes. Objective: The goal of this study was to investigate the role of Cygb in cardiomyocytes and identify the molecular pathways underlying its cytoprotective function. Method and Results: Utilizing transgenic mice with cardiac specific overexpression of Cygb (TgCygb), we demonstrated that Cygb overexpression promoted cardiomyocyte proliferation as compared to the control hearts (NTg) (phospho-Histone 3 IHC: 17 ± 0.7 vs 7 ± 0.5 positive cells/mm 2 TgCygb vs NTg; p<0.05, n=3) . The enhanced cellular proliferation in TgCygb hearts was associated with larger hearts at baseline. However, with exposure to acute cardiac stress (subcutaneous Angiotensin II infusion), TgCygb hearts showed significantly reduced cell death, oxidative stress and an overall lower induction of the DNA damage response pathway as compared to the NTg hearts [TUNEL: 7 ± 0.8 vs 10 ± 1.2 TUNEL positive nuclei/mm 2 , p<0.05, n=3; phospho-ATM IHC: 11 ± 2 vs 22 ± 1.5 phospho-ATM positive cells/mm 2 , p<0.05, n=5, dihydroethidium (DHE) staining: 78 ± 0.6% vs 98 ± 0.7% DHE positive nuclei/mm 2 , p<0.05, n=5]. Using single cell electrophoresis (Comet assay), we demonstrated that Cygb overexpressing cardiomyocytes underwent significantly lower DNA damage due to single and double stranded DNA breaks following exposure to oxidative stress as compared to controls cardiomyocytes (36 ± 2.1 vs 48 ± 3.1; p<0.05, n=6). Finally, chromatin immunoprecipitation, quantitative PCR and immunoblotting indicated that Cygb activates the expression of apurinic/apyrimidinic endonuclease 1 ( Ape1 ) and thus promotes DNA base excision repair following acute cardiac injury. Conclusions: Our data demonstrate that Cygb promotes cellular proliferation and protects cardiomyocytes against oxidative stress-mediated DNA damage by directly activating Ape1 expression.

Effects of Dietary Arginine Supplementation in Pregnant Mares on Maternal Metabolism and Foal Birthweight

Withers height is an important economic trait for the American Miniature Horses (AMHs) as the breeders' primary objective is to produce small and proportionate animals. Identification of Quantitative Trait Loci (QTLs) affecting height in this breed will result in a better understanding of the genetic architecture and biological pathways contributing to skeletal development. In earlier work, we genotyped 48 horses from 16 different breeds that represent extremes in body size, using the Equine SNP50 bead chip (Illumina Inc). In this study, we applied a dominant model Genome-wide association study (GWAS), a genome-wide fixation index (FST) estimation, as well as a cross-population composite likelihood ratio (XP-CLR) test between the eight large and eight small breeds from this data set. Our GWAS implicated the marker at ECA1:37,676,322 bp, positioned within an intron of the ANKRD1 gene, was significantly associated with height variation. An XP-CLR scan further supported a role in height for the ECA1:37,676,322 bp locus and the FST analysis supported correlations to IGF2BP2 at ECA19:23,815,750. To confirm our findings, we genotyped the candidate loci in an independent set of 89 AMHs. This set verified that the locus at ANKRD1 gene indeed correlates to height under a dominant mode of inheritance, but not the locus at the IGF2BP2 gene. ANKRD1, a transcription factor involved in myocyte growth and differentiation, may contribute to height by influencing the overall growth of the horses. This marker will be a valuable tool for the selection of breeding stock in breeds with height restrictions for registration.

Effect of restriction vegan diet's on muscle mass, oxidative status, and myocytes differentiation: A pilot study.

This study was conceived to evaluate the effects of three different diets on body composition, metabolic parameters, and serum oxidative status. We enrolled three groups of healthy men (omnivores, vegetarians, and vegans) with similar age, weight and BMI, and we observed a significant decrease in muscle mass index and lean body mass in vegan compared to vegetarian and omnivore groups, and higher serum homocysteine levels in vegetarians and vegans compared to omnivores. We studied whether serum from omnivore, vegetarian, and vegan subjects affected oxidative stress, growth and differentiation of both cardiomyoblast cell line H9c2 and H-H9c2 (H9c2 treated with H2 O2 to induce oxidative damage). We demonstrated that vegan sera treatment of both H9c2 and H-H9c2 cells induced an increase of TBARS values and cell death and a decrease of free NO2- compared to vegetarian and omnivorous sera. Afterwards, we investigated the protective effects of vegan, vegetarian, and omnivore sera on the morphological changes induced by H2 O2 in H9c2 cell line. We showed that the omnivorous sera had major antioxidant and differentiation properties compared to vegetarian and vegan sera. Finally, we evaluated the influence of the three different groups of sera on MAPKs pathway and our data suggested that ERK expression increased in H-H9c2 cells treated with vegetarian and vegan sera and could promote cell death. The results obtained in this study demonstrated that restrictive vegan diet could not prevent the onset of metabolic and cardiovascular diseases nor protect by oxidative damage.

Reduced fibre size, capillary supply and mitochondrial activity in constitutional thinness' skeletal muscle.

Constitutional thinness (CT) is a rare condition of natural low body weight, with no psychological issues, no marker of undernutrition and a resistance to weight gain. This study evaluated the skeletal muscle phenotype of CT women by comparison with a normal BMI control group. Ten CT women (BMI<17.5kg/m2 ) and 10 female controls (BMI: 18.5-25kg/m2 ) underwent metabolic and hormonal assessment along with muscle biopsies to analyse the skeletal muscular fibres pattern, capillarity, enzymes activities and transcriptomics. Constitutional thinness displayed similar energy balance metabolic and hormonal profile to controls. Constitutional thinness presented with lower mean area of all the skeletal muscular fibres (-24%, P=.01) and percentage of slow-twitch type I fibres (-25%, P=.02, respectively). Significant downregulation of the mRNA expression of several mitochondrial-related genes and triglycerides metabolism was found along with low cytochrome c oxidase (COX) activity and capillary network in type I fibres. Pre- and post-mitochondrial respiratory chain enzymes levels were found similar to controls. Transcriptomics also revealed downregulation of cytoskeletal-related genes. Diminished type I fibres, decreased mitochondrial and metabolic activity suggested by these results are discordant with normal resting metabolic rate of CT subjects. Downregulated genes related to cytoskeletal proteins and myocyte differentiation could account for CT's resistance to weight gain.

Expression and effects of TSP50 in mouse embryo and cardiac myocyte development

TSP50, a testis-specific gene encoding a serine protease-like protein, was specifically expressed in the spermatocytes of testes but abnormally activated and expressed in many different kinds of cancers. Here, we aimed to analyze the expression of TSP50 in mouse embryo and its function in early embryonic development. Firstly, the distribution of TSP50 in oocytes and embryonic development was characterized by immunofluorescence, RT-PCR and western blotting, and the results showed that TSP50 was detected at all studied stages with a dynamic expression pattern. When overexpressed TSP50 in zygotes by microinjection, the zygotes development was highly accelerated. On the contrary, knocking down TSP50 expression by RNA interference greatly retarded the zygote development. Furthermore, TSP50 expression at embryonic day 6.5 (E6.5), day 8.5 (E8.5) and day 10.5 (E10.5) were increasingly enhanced, However, the expression of TSP50 decreased gradually in the development and differentiation of cardiac myocyte from E12.5 to postnatal (P0).Additionally, we found that TSP50 expression was decreased during cardiac myocyte differentiation of P19 cells. Overexpression of TSP50 could decrease the expression of GATA-4, and knockdown of TSP50 markedly increase the expression of GATA-4. Taken together, our data indicate that TSP50 may play an important role during the process of mouse embryonic development as well as myocardial cell differentiation.

Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation.

Mature skeletal muscle cells cannot be expanded in culture systems. Therefore, it is difficult to construct an in vitro model for muscle diseases. To establish an efficient protocol for myogenic differentiation of human adipose tissue-derived stem cells (hADSCs), we investigated whether addition of IL-6 and/or myocyte-conditioned media (CM) to conventional differentiation media can shorten the differentiation period. hADSCs were differentiated to myocytes using the conventional protocol or modified with the addition of 25 pg/mL IL-6 and/or C2C12 CM (25% v/v). The expression of MyoD and myogenine mRNA was significantly higher at 5–6 days after differentiation using the modified protocol than with the conventional protocol. mRNA and protein expression of myosin heavy chain, a marker of myotubes, was significantly upregulated at 28 and 42 days of differentiation using the modified protocol, and the level achieved after a 4-week differentiation period was similar to that achieved at 6 weeks using the conventional protocol. The expression of p-STAT3 was significantly increased when the modified protocol was used. Similarly, addition of colivelin, a STAT3 activator, instead of IL-6 and C2C12 CM, promoted the myogenic differentiation of ADSCs. The modified protocol improved differentiation efficiency and reduced the time required for differentiation of myocytes. It might be helpful to save cost and time when preparing myocytes for cell therapies and drug discovery.

Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

ABSTRACTAmong rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.

The influence of IGF-Ⅰ on the expression of Myf5 and TGF-β1 in medial rectus muscle of cat model with strabismus

Objective: To observe the influence of exogenous insulin-like growth factor-Ⅰ(IGF-Ⅰ) on the expression of myocyte differentiation factor 5 (Myf5) and transforming growth factor β1(TGF-β1) in medial rectus muscle of cat model with strabismus. Methods: Experiment research. Twenty-seven kittens which were in sensitive period of visual development (4-6 weeks old), were randomly divided into experimental, control and blank control groups by random numbers table method. Each experimental group was further divided into 3 sub-groups (4 weeks, 8 weeks and 12 weeks) based on drug intervention time, hence 3 kittens in each sub-group. The control group and the blank control group were also divided into 3 sub-groups respectively. Exotropia treatment models were set up through surgical methods and injection of IGF-Ⅰ(0.05 ml,0.1 g/L). The internal rectus muscles of 4 weeks sub-group, 8 weeks sub-group and 12 weeks sub-group were taken respectively after the treatment model had been set up. The internal rectus muscles of the control group and the blank control group were also taken according to corresponding time. The expressions of Myf5 and TGF-β1 were tested with immunohistochemistry staining method, and optical density analysis method were employed to measure the average optical density value. The expression of Myf5 and TGF-β1 was analyzed by Kruskal-Wallis and Bonferroni test. The correlation between the expression of Myf5 and TGF-β1 and the time of drug intervention was analyzed by simple linear regression. Results: (1) In the experimental group, the expression of the Myf5 of the 4 weeks sub-group, 8 weeks sub-group and 12 weeks sub-group were 33.34±17.16, 39.24±15.25 and 47.70±19.39, which were higher than the control group (21.30±7.44, 19.43±4.75, 4.82±2.66) and the blank control group (18.95±6.59, 18.00±7.29, 5.86±2.61) at the same time point, and the differences were statistically significant in 8 weeks sub-group and 12 weeks sub-group (χ(2)=21.864, 31.814, both P<0.01). The expression of Myf5 in the experimental group increased with the extension of IGF-Ⅰ intervention time (R(2)=0.99, P<0.05). But there were negative correlation between expression of Myf5 and drug intervention times in the control group and the blank control group (R(2)=0.81, 0.80, both P<0.05). (2) In the experimental group, the expression of the TGF-β1 of the 4 weeks sub-group, 8 weeks sub-group and 12 sub-weeks group were 0.80±0.12, 0.53±0.09, 0.42±0.08, which were higher than the control group (1.91±0.23, 2.30±1.03, 1.82±0.72) and the blank control group (2.01±0.31, 2.62±1.11, 1.83±0.67) at the same time point, and the differences were statistically significant (χ(2)=30.801, 40.278, 35.177, all P<0.01). The expression of TGF-β1 in the experimental group decreased with the extension of IGF-Ⅰintervention time (R(2)=0.83, P<0.05). The average optical density value regression equation of TGF-β1 in the sterile water control group and the blank control group was 0.04 and 0.06, respectively, and the fitting degree was very poor. Therefore, there was no correlation trend with time. Conclusions: Exogenic IGF-Ⅰ could enhance the expression of Myf5 in medial rectus muscle of cat model with strabismus. Exogenic IGF-Ⅰ could inhibit the expression of TGF-β1 in medial rectus muscle of cat model with strabismus. Repeated injection of exogenous IGF-Ⅰ may continuously enhance the expression of Myf5 and inhibit the expression of TGF-β1. (Chin J Ophthalmol, 2018, 54: 375-382).

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.

Regulatory mechanism for lncRNAs in skeletal muscle development and progress on its research in domestic animals

Skeletal muscle is an essential tissue to maintain the normal functions of an organism. It is also closely associated with important economic performance, such as carcass weight, of domestic animals. In recent years, studies using high-throughput sequencing techniques have identified numerous long non-coding RNAs (lncRNAs) with myogenic functions involved in regulation of gene expression at multiple levels, including epigenetic, transcriptional and post-transcriptional regulation. These lncRNAs target myogenic factors, which participate in all processes of skeletal muscle development, including proliferation, migration and differentiation of skeletal muscle stem cells, proliferation, differentiation and fusion of myocytes, muscle hypertrophy and conversion of muscle fiber types. In this review, we summarize the functional roles of lncRNAs in regulation of myogenesis in humans and mice, describe the methods for the analysis of lncRNA function, discuss the progress of lncRNA research in domestic animals, and highlight the current problems and challenges in lncRNA research on livestock production. We hope to provide a useful reference for research on lncRNA in domestic animals, thereby further identifying the molecular regulatory mechanisms in skeletal muscle growth and development.

Enhancer epigenomic regulation in differentiation, development and cancer

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We previously identified CBP/p300 as major H3K27 acetyltransferases and MLL3/MLL4 as major enhancer H3K4me1 methyltransferases [1, 2]. We show that during differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 are required for enhancer activation, cell type‐specific gene expression, and cell differentiation [2, 3]. Further, we show that while enhancer‐priming by MLL3/MLL4 is dispensable for cell identity maintenance, it controls cell fate transition by orchestrating CBP/p300‐mediated enhancer activation. MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers [3, 4].Our data suggest a model of sequential actions of epigenomic regulators on enhancers: 1) pioneer TFs and lineage‐determining TFs recruit MLL3/MLL4 to prime enhancer regions and label them with H3K4me1; 2) MLL3/MLL4 facilitate the binding of CBP/p300, which activate enhancers and label them with H3K27ac; 3) H3K27ac and acetylated TFs are recognized by the epigenomic reader Brd4, which recruits Mediator and RNA Pol II to establish enhancer‐promoter interactions and activate cell type‐specific genes (Nat Comm, in press).MLL3/MLL4 are tumor suppressors frequently mutated in many types of cancers as well as developmental diseases. We show that MLL3/MLL4 are required for the development of adipose, muscle, mammary gland, B cells, T cells, and heart and that depletion of MLL3/MLL4 in mice promotes tumorigenesis [2, 5–9]. Our findings suggest that mutations in MLL3/MLL4 would lead to defects in enhancer activation, cell type‐specific gene expression and cell differentiation. Such a mechanism may contribute to the pathogenesis of these developmental diseases and cancers.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIDDK, NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Cardiac Troponin‐T in Cardiac H9c2 Cells During Co‐Culture with Mesenchymal Stem Cells

Previous studies have shown that intravenous injection of mouse bone marrow mesenchymal stem cells (MSC) attenuate the loss of function in the heart after coronary artery occlusion (Boomsma et al, Int J Cardiol 122:17 2007) and secrete paracrine factors that may be responsible for their beneficial effect (Boomsma and Geenen, PLoSONE 7:e3685, 2012). Mouse MSC were cultured in Mesencult + 20% serum supplement (Stem Cell Technologies) and rat cardiac H9c2 cells were cultured in DMEM + 10% FBS until confluent. MSC and H9c2 cells were then combined equally and plated in 6‐well dishes at a total concentration of 1.0 ×106 cells/well in DMEM + 10% FBS. After 2 days the media was changed to DMEM + 1% heat inactivated horse serum to promote myocyte differentiation. Control cultures containing H9c2 cells alone at the same cellular concentrations were treated similarly. After 1, 3, 5, and 7 days of culture in 1% serum RNA was isolated using RNAqueous 4 PCR (Ambion) and treated with DNAse. Cardiac troponin‐T (cTnT) was quantified in H9c2 cells by RT‐qPCR using an iScript cDNA Synthesis Kit and iQ SYBR Green Mastermix (BioRad). Relative expression was determined using the reference gene GAPDH; no‐RT and water controls were included. Species specific primers for rat cTnT and rat GAPDH were designed in order to separate out expression of cTnT in H9c2 cells (rat) from MSC (mouse). Mean and standard error was calculated for each group and significance was determined using Student's t‐test (p&lt;0.05, n=5–6). Relative expression of cTnT in H9c2 cells was low in both co‐culture (0.422 ± 0.157) or alone (0.427 ± 0.065) after 1 day in 1% serum, but increased more than 10‐fold after 3–7 days in both co‐culture or alone. cTnT expression in H9c2 cells was significantly greater after 5 days in co‐culture with MSC (4.204 ± 0.524) compared to H9c2 cells cultured for 5 days alone (2.353 ± 0.510; p&lt;0.05). Expression in H9c2 cells cultured alone continued to increase after 7 days (5.958 ± 1.179) such that there was no statistical difference from cells in co‐culture (3.918 ± 1.880). This study demonstrates that MSC accelerate cTnT expression in H9c2 cells and may partially explain the beneficial effect of MSC on cardiac function in vivo.Support or Funding InformationSupported by a VanderVelde Research Scholarship and a Summer Research Grant from Trinity Christian College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Micro‐RNA‐205 Regulates Heart Size Through Direct Modulation of the Hippo Pathway

During fetal development, the mammalian myocardium undergoes a period of hyperplastic growth, which establishes the number of cardiomyocytes in the adult heart. After birth, cardiomyocytes proceed through a final round of cell division in the absence cytokinesis that results in binucleation of &gt;95% of adult cardiomyocytes. Nearly all subsequent increase in myocardial mass is due to myocardial hypertrophy, with extremely low number of new cardiomyocytes being produced throughout post‐natal life. Despite the importance of this phenomenon, little is known about the molecular/genetic basis, especially with regard to the role of micro‐RNAs, for the transition from hyperplastic to hypertrophic‐based myocardial growth.We hypothesize a specific perinatal heart micro‐RNA‐mediated gene program is necessary for the normal transition from a fetal heart to an adult heart gene program.To identify the molecular mechanisms and genetic pathways involved in cardiac myocyte differentiation, RNA was isolated from E19, and 1, 3, 5, 7, 10 and 35‐day old mouse hearts (n=9 hearts/time point pooled). Cardiomyocyte micro‐RNA profiles (n=3 arrays/time point) were measured and bioinformatic analysis was used to identify genes that are transiently and significantly changing (p&lt;0.05, fold change &gt;1.5) during the perinatal period.Our analysis identified microRNA‐205 as a candidate for playing a role in the cardiac transitional program. Previous studies have shown a global knockout of miR‐205 to be neonatally lethal. We observe a transient 20‐fold increase in miR‐205 expression between day 1 and day 5 of post‐natal life, with levels returning to baseline by day 10. In‐situ hybridization revealed miR‐205 expression to be restricted to the epicardium of the heart.Mice harbouring a cardiomyocyte‐specific deletion of miR‐205 using αMHC‐Cre are born healthy with expected Mendelian ratios, and develop through the neonatal period normally. Hearts collected from adult mice show signs of abnormal growth and hypertrophy, up to 50% larger than controls. Previous studies demonstrated that miR‐205 directly targets YAP within the evolutionarily conserved hippo pathway that controls organ size. Hearts lacking miR‐205 exhibit a substantial increase in YAP protein expression. Increased cardiac size has been demonstrated in a constitutively active YAP transgenic mouse model. We conclude that miR‐205 plays a direct role in regulating post‐natal heart size through direct modulation of the Hippo pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of LncRNA expression in cell differentiation

ABSTRACTLineage-specific cell differentiation is a precise and coordinated biological process. To explore the roles of long noncoding RNA (lncRNA) in this process, the expression of polyA-minus RNAs was comparatively studied during the course of myocyte and adipocyte differentiation. In addition to identifying thousands of novel lncRNAs, distinct lncRNA profiles were revealed during lineage-specific differentiation, showing their active involvement in this process. This study further found that lncRNAs were organized in clusters and are co-regulated, constituting transcription open domains (TODs). In myocyte differentiation of C2C12 cells, loss-of-function screening identified three myogenic lncRNAs. Knockdown of their expression compromised not only the differentiation process, but also the essential signaling pathway. In addition to showing that lncRNAs are actively involved in cell differentiation, our results start to reveal a comprehensive signaling pathway, involving both protein and lncRNA factors.

Vangl2 is essential for myocardial remodeling activated by Wnt/JNK signaling

The Wnt/JNK pathway, responsible for tissue polarity in cardiogenesis in vertebrates, has been shown to play numerous roles during differentiation and development of cardiac myocytes. Van Gogh-like-2 (Vangl2) is a core component that regulates the induction of polarized cellular and tissue morphology during animal development. However, little is known about Wnt/JNK signaling pathway in the process of myocardial remodeling. In present study, we found that activation of Wnt/JNK signaling by Wnt5a stimulates enlargement of cardiomyocyte surface area. The hypertrophic features were inhibited in Vangl2 depleted cells. Meanwhile, Wnt/JNK activation induced cytoskeleton rearrangement but failed to activate these effects in cells lacking Vangl2. Moreover, Wnt/JNK activation significantly increased the cell apoptosis by mediating the mitochondrial permeability transition pore (mPTP) dysfunction, whereas knockdown of Vangl2 partly reversed these effects. These results suggest that activation of Wnt/JNK signaling stimulates myocardial remodeling (cell morphological changes, apoptosis and mitochondrial dysfunction), in which Vangl2 may play an essential role.

Neonatal Permeability Transition Pore Closure is Associated with Increased Cardiac Function

Introduction: Recent studies have shown that embryonic myocytes have less active mitochondria and immature cellular structure. The neonatal heart is also closer in structure and function to the fetal heart, but progressively differentiates into its mature form with time. Mitochondria appear to regulate these changes during the neonatal transition. The closure of the mitochondrial permeability transition pore (PTP), by pharmacologic or genetic inhibition of Cyclophilin D (CyPD), is associated with differentiation in cultured neonatal myocytes. We evaluated CyPD inhibition's effect on in vivo cardiac function. We hypothesized that closure of the mPTP in the neonatal mouse heart will increase cardiac function by enhancing myocyte differentiation and maturation without longer-term deleterious effects. Methods: One-day-old WT or CyPD null mice were treated with daily intraperitoneal injections of vehicle (inert), Cyclosporin A (CsA), or NIM811 for five days. At six days old and weanling, the mice underwent echocardiography to evaluate ejection fraction (EF), a measure of cardiac function. Hearts were then harvested for further biochemical analysis.Results: WT mice hearts treated with CsA or NIM811, as well as CyPD nulls, had significantly higher EF at six-days-old (P<0.0001) compared to WT untreated or vehicle-treated mice. At weanling, there was no significant difference in EF across all groups and all survived to this time point. Work regarding assays of electron transport chain function and structure remains ongoing. Conclusion: Our results demonstrate that PTP closure in the neonatal mouse heart is associated with increased EF, as compared to vehicle treated mice. Our most recent data suggests that there is no significant difference in EF at weanling across all groups. This suggests that PTP closure enhances the neonatal transition to a more differentiated myocardium leading to an early increase in ejection fraction usually observed in mature animals.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.