skip to main content
Explore Scholarly Publications and Datasets in the NSF-PAR

  • NSF Public Access
  • Search Results
  • Accepted Manuscript: Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply
Title: Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply
Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B 12 ) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, more » the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest ( p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism. « less
Authors:
; ; ; ; ; ; ; ;
Award ID(s):
2019077
Publication Date:
NSF-PAR ID:
10357570
Journal Name:
Frontiers in Genetics
Volume:
13
ISSN:
1664-8021
Sponsoring Org:
National Science Foundation
More Like this
  1. ( , Circulation)
    Introduction: Myocardial fibrosis and dysfunction is one of the major cardiac complications of long-term diabetes. Prolonged hyperglycemia is known to induce myocardial dysfunction often leading up to heart failure. Hypothesis: The objective of this study was to investigate the cardioprotective effect of glycyrrhizin (GLC) on myocardial damage in engineered in-vitro human cardiac tissues. Engineered 3D tissue chips present an ideal microenvironment via therapeutically relevant interfaces to study molecular- and cellular-level events and mimic human-specific disease states, and identify new therapeutic targets in vitro. Methods: AC16 human cardiomyocyte cells were used to 3D bioprint cardiac tissue chips based on prior published work. In our study, the 3D bioprinted cardiac tissue chips (CTC) were cultured using normo- (5mM) and hyper-glycemic (25mM) conditions for up to 48 hrs. For the GLC treatment group, a subset of CTC cultured using hyperglycemic conditions were treated with 50 mM of GLC for 24 hours. Results: CTC cultured under hyperglycemic conditions demonstrated altered levels of connexin-43 (CX43) and Troponin-I implying cardiomyocyte injury. Exposure to hyperglycemia revealed changes in epigenetic markers: histone methylation marker (H3K9me)-1, Sirtuin-1, and Histone Deacetylase (HDAC)-2 as well as in inflammatory and stress related mediators such as heat shock protein (HSP)-60, receptor for advancedmore »glycation end products (RAGE), toll like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. CTC exposed to 25mM glucose for 24 hours resulted in the downregulation of HSP60 and Sirtuin-1. Prolonged exposure to hyperglycemia led to decrease in the expression of CX43 and CXCR4; thereby adversely affecting cardiomyocyte function. Upregulated expression of DNA-binding nuclear protein HMGB1 along with changes in H3K9me1 indicated long-term hyperglycemia-induced damage to cardiomyocytes. GLC treated CTC exhibited a decrease in the expression of RAGE, TLR4 and also demonstrated altered expression of CX43, CXCR4, and troponin I. Conclusions: This study suggests that GLC possesses cardioprotective effects in human cardiomyocytes exposed to prolonged hyperglycemia.« less
  2. ; ; ; ; ; ; ; ; ; ( , American Journal of Physiology-Cell Physiology)
    Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migrationmore »is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.« less
  3. ( , Circulation research)
    Type-II diabetes (T2D) patients affected by underlying hyperglycemic (high glucose/blood sugar) conditions often suffer from cardiac atrophy, resulting in tissue mass reduction and debilitating cardiac health. To understand pathophysiological mechanisms during progression of cardiac atrophy, a 3D bioprinted organoid platform was developed from a mixture of hydrogels containing human cardiac cells, including cardiomyocytes (CM), fibroblasts (CF) and endothelial cells (EC), to mimic the functionality of the in-vivo tissue. The organoids were cultured using normoglycemic- or hyperglycemic-conditions. The expression of essential biomarkers in these organoids, for myocardin (Myocd), troponin-I (TRP-I), fibroblast protein-1 (FSP-1) and endothelin-1 (ET-1) was confirmed. To assess the physiological cellular connections during hyperglycemia, the presence of Connexin-43 (CX-43) was assessed in the presence of a CX-43 blocker, gap26. Epigenomic tools were used to simultaneously interrogate histone-modifications by histone 3 lysine 9 mono-methylation (H3K9me1) along with the co-regulation of inflammatory mediators, such as the high mobility group box 1 (HMGB1) and toll like receptor 4 (TLR4) in the cardiac organoids cultured using normal versus hyperglycemic conditions. Organoids exposed to high glucose showed an increased expression of H3K9me1 as well as inflammatory mediators HMGB1 and TLR4. Hyperglycemia also exhibited alterations in expression of Myocd and FSP-1 in the organoids, comparedmore »to normoglycemic conditions. Treatment with gap26 affected the CX-43 expression significantly, in organoids cultured under hyperglycemia suggesting that high glucose conditions associated with prolonged diabetes may lead to compromised CM-CF coupling, essential for maintenance of cardiac functionality. Increased levels of H3K9me1 suggest decreased expression of Myocd, which may lead to CM degeneration. Epigenetic modifications including alterations in histone methylation in regulation of the myocardial genes and gap junction proteins under hyperglycemic conditions, may lead to cardiac atrophy. We expect to establish an actual T2D patient iPSC cell derived cardiac platform, to offer new therapeutic opportunities within the field.« less
  4. ; ; ; ; ( , Clinical Epigenetics)
    Abstract Background Mutations in LMNA , encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. Results Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA -related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutationsmore »were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. Conclusions Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.« less
  5. ; ; ; ; ; ; ; ( , Epigenetics & Chromatin)
    Abstract Background

    Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species.

     Results

    Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such asSCN5Amay account for differences in heart rate, while genes such asTNNT2andTPM3maymore »underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated.

     Conclusions

    Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

    « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email