All experimental methods were carried out in agreement with applicable international, national, and institutional guidelines for the care and use of animals (Animal Welfare Act and National Institutes of Health policies) and were approved by the Sanford Research Institutional Animal Care and Use Committee. Sprague Dawley rats (Harlan Laboratories, Indianapolis, IN) were used in all experiments and housed in Sanford Research’s Animal Resource Center, a climate-controlled, light-dark cycled facility.
Animal model characteristics
Methods and model characteristics of the four animal groups used in this study have been detailed previously [29, 30]. Briefly, young adult female rats received either control or HF diet (Teklad, Harlan Laboratories, Madison, WI) for at least 28 days before mating and throughout pregnancy. Gestational day zero (GD0) was determined by a positive vaginal swab for spermatozoa. On GD14, after confirmation of pregnancy through ultrasound, dams received either citrate buffer (0.09 M) or 65 mg/kg of intraperitoneal streptozotocin (Sigma Life Sciences, St. Louis, MO) to induce diabetes in the last third of pregnancy. Hyperglycemia was partially controlled with twice daily insulin treatments to keep glucose levels in a target range of 200-400 mg/dl. Dams that received streptozotocin but did not manifest a fasting blood glucose level ≥ 200 mg/dl were excluded from the study. Delivery (~ GD22) yielded postnatal day one (P1) offspring from four distinct groups: exposed to maternal diabetes alone, exposed to maternal HF diet alone, exposed to the combination of both maternal diabetes and HF diet, and control group (Figure 1A). We used all four groups for gene expression analysis with further experiments streamlined to control and combination exposed groups.
Total RNA isolation and quantification
Hearts were extracted from the following four experimental groups of newborn P1 rats: control, diabetes exposed, HF diet exposed, and combination exposed. Immediately after harvesting, samples were snap frozen in liquid nitrogen, and stored at -80oC until RNA extraction. Each experimental group consisted of a pool of male and female rat hearts. Total RNA was extracted from the whole heart with TRIzol and purified using an affinity resin column (Qiagen RNeasy Mini kit, Germantown, MD) according to manufacturer’s protocol. Total RNA concentration was performed using spectrophotometric analysis measurement (abs-emission A260/A280) using NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc. Waltham, MA). RNA sample integrity was assessed by electropherogram analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and only samples with RNA integrity number (RIN) scores > 8 were used for microarray labeling and hybridization.
Microarray hybridization and data analysis
Microarray hybridization was performed by the Analytical Genomics Core Facility (Sanford Burnham Medical Discovery Institute, Lake Nona, Orlando, FL) using GeneChip Rat Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA) according to manufacturer’s protocol. Briefly, total isolated RNA (100 ng) from each sample was converted to cDNA utilizing SuperScript III First Strand Synthesis Supermix (Invitrogen, Life Technologies Corporation, Carlsbad, CA). Labeled complimentary RNA (cRNA), synthesized and amplified from the double-stranded cDNA template, was fragmented and hybridized onto GeneChip arrays. As a measure of quality control of the fragmented biotin-labeled cRNA, a prior hybridization of a test-3 array was performed and analyzed. GeneChip 3000 scanner (Affymetrix, Santa Clara, CA) was used to scan and quantitatively analyze images of hybridized GeneChip arrays. Intensity values for each probe cell in the arrays were calculated by GeneChip software.
Data normalization and analysis was performed using GeneSpring GX 14.01 (Agilent Technologies, Palo Alto, CA). Probe cell intensities were used to calculate an average intensity for each set of probe pairs representing a gene. Quality control (QC) filtering was performed on the normalized intensity values and entities were clustered into four conditions: diabetes, HF diet, combination exposed and controls. Gene expression profiles for each condition were visualized as volcano plots to identify genes significantly upregulated or downregulated in each group.
Bioinformatics analysis
Gene ontology deconvolution and functional annotation analysis
Statistically significant gene expression profiles from each comparison were separated into upregulated and downregulated lists for functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources v6.8 (https://david.ncifcrf.gov/). To determine over representation or enrichment, the DAVID algorithm employs a modified Fisher’s exact test that is incorporated into a score that reports relative priority. Distinct gene lists for each comparison were submitted to DAVID using Entrez Gene identifiers for functional annotation analysis. A high classification stringency was selected to maintain robust groups, and scores were reported for KEGG and Reactome pathways when applicable. Further functional pathway analysis of a given gene list was done through Reactome pathways database analysis tool (Reactome v69; https://reactome.org/).
Functional networks analysis and gene targets prioritization
Ingenuity pathway analysis (IPA; Qiagen, Germantown, MD) was performed to map functional gene networks defined by the quality-filtered transcriptome. Highest priority network scores were determined and all the gene relationships, i.e. functional interactions among genes, were exported from IPA for use in Cytoscape v3.7.1 (https://cytoscape.org/) for further network analysis. Prioritization of gene targets was achieved through graph theory analysis tools within Cytoscape. Molecule Activity Predictor Analysis module in IPA was used to predict activation or inhibition of non-focused neighboring molecules, defined by IPA as molecules not included in the uploaded list of genes/molecules, within the functional network. This prediction analysis is based on the expression of the focused molecules, also known as statistically significant genes, within the network and predicts either upstream and/or downstream activities.
Determination of mitochondrial-associated genes
MitoCarta 2.0 database (Broad Institute, Cambridge, MA) was used to determine the mitochondrial-associated genes in our list of statistically significant genes. MitoCarta 2.0 is an online repository of 1158 mammalian (human and mouse) genes encoding proteins where their mitochondrial localization has been validated by various methods. We cross-referenced data from MitoCarta with data from the current study to identify mitochondrial-related genes in our gene expression dataset. The recently updated mouse MitoCarta 2.0 database used in the present analysis can be found at the following website: (https://www.broadinstitute.org/files/shared/metabolism/mitocarta/mouse.mitocarta.2.0.html).
Quantitative RT PCR
RNA was extracted from newborn (P1) rat hearts using the RNeasy Fibrous Tissue Mini kit (Qiagen, Germantown, MD) following manufacturer’s protocol. RNA integrity was assessed by electropherograms using 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA) and demonstrated RIN scores of 9.2-10 (average=9.8). RNA concentration from two groups, control and combination exposed, was measured by Epoch spectrophotometer (BioTek, Winooski, VT). Complementary DNA (cDNA) was synthesized using iScript cDNA Synthesis Kit and T100 Thermal Cycler (Bio-Rad, Hercules, California). Quantitative PCR (qPCR) was performed by TaqMan Gene Expression Assays approach with ABsolute Blue QPCR Mix (Thermo Fisher Scientific, Waltham, MA) using an ABI7500 qPCR system (Thermo Fisher Scientific, Waltham, MA). Beta-2-microglobulin (B2m) was used as the reference gene. Cardiac expression relative to B2m was compared between the control and combination exposed groups (n = 6 males/group). B2m, mitochondrial ribosomal protein L19 (Mrpl19), mitochondrial ribosomal protein S27 (Mrps27), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc1a) and fibroblast growth factor receptor 2 (Fgfr2) probe/primer sets were obtained from Thermo Fisher Scientific (Waltham, MA), and death associated protein 3 (Dap3) probe/primer set was obtained from Integrated DNA Technologies (Coralville, IA).
Western blot analysis
Newborn (P1) rat hearts from control and combination exposed males were homogenized and sonicated in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) with cOmplete protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA) and Cytation 3 Spectrophotometer (BioTek, Winooski, VT). Protein (20 µg) was prepared using Laemmli buffer and reducing agent then subjected to electrophoresis on 4-15% Criterion TGX Gels using Tris/Glycine/SDS buffer (Bio-Rad). MagicMark XP Western Protein Standard (Thermo Fisher Scientific, Waltham, MA) was used to identify band size. Gels were transferred to PVDF membranes using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were dried, rehydrated in methanol, washed in TBS, blocked in TBS containing 10% Clear Milk Blocking Buffer (Thermo Fisher Scientific) and then incubated overnight at 4ºC with primary antibody. After washing in TBS-T, membranes were blocked again and incubated with secondary antibody for 1 hr, using goat anti-rabbit IgG-HRP for reference proteins (Southern Biotech, Birmingham, AL) or donkey anti-rabbit IgG IRDye 680RD (LI-COR, Lincoln, NE) for proteins of interest. HRP exposed bands were visualized using Luminata Forte HRP Chemiluminescence Substrate (Thermo Fisher Scientific). Images were captured using a ChemiDoc MP Imaging System (Bio-Rad) and densitometric analysis was done using ImageJ. Optical density (OD) measurements from tested proteins were normalized to primary reference protein β-ACTIN. Voltage-dependent anion channel or porin (VDAC) and translocase of outer mitochondrial membrane 20 (TOMM20), both outer mitochondrial membrane proteins, were used as secondary references.
Enzymatic assays
Insulin and c-peptide levels were measured on 25ul of newborn (P1) serum using the MILLIPLEX Map Rat Metabolic panel (MilliporeSigma, Burlington, MA) as previously done [26, 29] and Total Protein kinase B isoform 2 (AKT2) and Ser473-phosphorylated AKT2 (regulatory site for insulin signaling) were measured using the MILLIPLEX Map Phospho/Total AKT2 2-plex Magnetic Bead Panel (MilliporeSigma, Burlington, MA) according to manufacturer’s protocol as described [29]. Briefly, 15ug of aforementioned newborn (P1) rat heart protein was incubated overnight with antibody coated beads. The beads were then washed and incubated with Detection Antibody for 1hr. Streptavidin-PE was then used as a reporter molecule. The plates were read and analyzed using Luminex 200 Milliplex Analyzer (MilliporeSigma, Burlington, MA).
Total glycogen synthase kinase 3 beta (GSK3β) and Ser9 phosphorylated GSK3β were measured from aforementioned newborn (P1) rat heart protein using GSK3β (Total/Phospho) Multispecies InstantOne ELISA Kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s protocol. In short, 30 mg of protein lysate was added to the ELISA plate and incubated with Antibody Cocktail for 1 hr. The wells were then washed with Wash Buffer and exposed to Detection Reagent. After 15 min, Stop Solution was added and the plate was read at 450 nm using Cytation 3 Spectrophotometer (BioTek, Winooski, VT).
Extracellular Flux (XF) analyses
A glycolytic stress test (GST) was used to compare basal and maximal glycolysis and reserve capacity of primary isolated neonatal (P1) rat cardiomyocytes (NRCM) from control and combination exposed offspring on Seahorse XF24 analyzer (Agilent Technologies, Palo Alto, CA). Methods and experimental validation were similar to that previously described [26]. Briefly, newborn hearts were harvested in ice-cold HBSS. Atria were removed and ventricles were minced then digested with 2 mg/mL DNase I and 0.15% Trypsin. Digestion was stopped with bovine serum (BS) and cells were pelleted and transferred to 10:1 DMEM-1 (DMEM with 10% BS and 1% penicillin/streptomycin) with DNase I. Cells were incubated on an uncoated plastic dish for 1 hr at 37°C in 5% CO2 to remove rapidly adhering fibroblasts. Live NRCM were then counted and plated at a seeding density of 40,000 cells/well to 0.1% gelatin coated V7-PS 24-well microplates in DMEM-2 (DMEM-1 with 100 µM bromodeoxyuridine) and incubated overnight (12 hr) at 37°C with humidified 5% CO2. The following morning, media was changed to XF base media (Agilent Technologies), incubated without CO2 for 1 hr, and then oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured at baseline and following injections to yield final well concentrations. Conditions were as follows:
- Port A: 10 mM D-(+)-glucose (Sigma G8644)
- Port B: 2 μM rotenone (Sigma R8875) + 4 μM antimycin A (Sigma A8674), respiratory complex inhibitors used to drive anaerobic glycolysis
- Port C: 200 μM monensin (Sigma M5273) + 0.25 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, Sigma C2920) which uncouples aerobic respiration to ensure attainment of maximal anaerobic glycolysis
Outcome measures included: baseline glycolysis, glycolysis following glucose injection, and maximal glycolysis (defined as peak ECAR following rotenone/antimycin) because FCCP/monensin did not usually increase ECAR further. The proton production rate (PPR) was calculated as originally described by Mookerjee et al. to discern acidification from anaerobic glycolysis vs mitochondrial respiration [26, 31].
Histopathology
Formalin fixed and paraffin embedded P1 rat hearts (n = 13) were sectioned and stained with periodic acid-Schiff (PAS) stain to qualitatively compare glycogen stores. In brief, biventricular cross-sections were deparaffinized and rehydrated, stained with 0.5% periodic acid for 5 minutes then Schiff solution for 15 minutes and washed in running water 10 for minutes. Sections were then counterstained with hematoxylin, dehydrated, and mounted with a coverslip. Sections were digitally imaged with the Aperio VERSA 8 automated slide scanner and qualitatively analyzed using the Aperio Image Scope Software (Leica Biosystems Imaging, Buffalo Grove, IL).
Mitochondrial copy number
Total DNA was extracted from P1 whole hearts (n=6-7 males/group) via DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD) following manufacturer’s instructions. DNA integrity and concentrations were determined using Epoch spectrophotometer (BioTek, Winooski, VT). Relative mitochondrial DNA copy number was determined using qPCR with primers designed for mitochondrial control region (D-loop; Integrated DNA Technologies, Coralville, IA) and cytochrome-c oxidase I (Mt-co1; Thermo Fisher Scientific, Waltham, MA) as previously described [26, 32]. All qPCR reactions were run in triplicate in ABsolute Blue QPCR Mix (Thermo Fisher Scientific) on a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA). Gene-specific standard curves were calculated using rat mitochondrial DNA and MxPro software (Agilent Technologies, Santa Clara, CA) and used to calculate relative mitochondrial DNA copy number.
Statistical analysis and data availability
Statistical analysis of the microarray gene expression was performed using the unpaired unequal variance t-test (Welch test). The hierarchical clustering for groups and entities was performed using Euclidean distance metric and Ward’s linkage algorithm. Statistical significance was set at fold change (FC) > 1.25 and p-value (p) < 0.05. The microarray datasets generated and analyzed in this study are available in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE150649. Results from PCR, protein expression, mitochondrial copy number and extracellular flux analyses were analyzed using student’s T-test or Mann-Whitney to detect significant differences between P1 male controls vs combination exposed offspring. These analyses were done using Prism software (GraphPad Software, La Jolla, CA) and statistical significance was set at p < 0.05.