Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice

We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER) gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA) based on the DNA microarray data from GPER-knockout versus GPER-intact (intact) cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article “Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling” (Wang et al., 2016) [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE86843.

specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling"   [1]. Data

Value of the data
This dataset provides the complete list of altered genes related to mitochondria and inflammatory response in GPER-knockout versus intact cardiomyocytes from mice of both sexes.
May facilitate further research that reveals the pathophysiology for sex-specific differences in heart disease.
May serve as a benchmark for comparison with data obtained from estrogen receptor (ER) α and ERβ cardiomyocyte-specific knockout mice for further insight into the functional roles of the estrogen receptors in the maintenance of cardiac structure and function.
May stimulate further research on the clinical potential of targeting GPER in the treatment of heart disease and other age-related disorders, in which mitochondrial dysfunction and inflammation have central roles in the underlying pathophysiology.

Data
To examine the differences in the mitochondrial and inflammatory response gene expressions between GPER-knockout and intact cardiomyocytes, microarray data were loaded into GSEA 2.0.1 software using GSEA gene sets "MITOCHONDRION (including 314 genes)" and "HALLMARK_IN-FLAMMATORY_RESPONSE (including 193 genes)" [1,2]. The altered individual mitochondrial and inflammatory genes in GPER knockout versus intact cardiomyocytes from both sexes are presented in Tables 1-4.

Cardiomyocyte isolation from GPER KO and GPER-intact or wild-type mice
Mice at 18-20 weeks of age were injected i.p. with 200 ml heparin (Sagent Pharmaceutical Inc., Schaumburg, IL, 100 IU/mouse) 10 min prior to anesthesia with pentobarbital (Akorn Inc., Lake Forest, IL, 100 mg/kg body weight) by i.p. injection. Upon verification of deep anesthesia by the absence of response to tail/toe pinches, the heart was quickly removed and trimmed in an ice-cold, calcium-free perfusion buffer (126 mM NaCl, 4.4 mM KCl, 1 mM MgCl2, 4 mM NaHCO3, 10 mM HEPES, 11 mM glucose, 30 mM 2,3-butanedine monoxime [Sigma, St. Louis, MO], 5 mM taurine [Sigma], pH 7.35). The heart was then cannulated through the aorta on an Easycell System for Cardiomyocyte Isolation (Harvard Apparatus, Holliston, MA) and perfused at 37°C with calcium-free perfusion buffer at a flow rate of 3 ml/min for 4-5 min until the effluent became clear. The heart was switched to digestion buffer (perfusion buffer plus 50 mM CaCl2 and 0.5 mg/ml collagenase II [Worthington Biochemical Corp., Freehold, NJ]), and perfused for 10-15 min at a flow rate of 4 ml/min until the heart was pale and flaccid. The heart was pulled from the cannula and the ventricles were transferred to a 60-mm sterile dish containing 5 ml of transfer buffer (perfusion buffer plus 0.1 mM CaCl2 and 2% bovine serum albumin [Sigma]) and cut into small pieces. The minced tissue was incubated in a 37°C water bath for 10 min. The cell suspension was filtered through a 100-μm mesh cell strainer (BD Biosciences, San Jose, CA) to remove tissue debris and spun at 420 rpm at room temperature for 2 min. After removing the supernatant, cardiomyocytes were washed with 1 ml of PBS and centrifuged at 1500 rpm at 4°C for 3 min. The cells were suspended in 1 ml of QIAzol (Qiagen Inc, Valencia, CA), mixed, and homogenized before storing at À 80°C.

DNA microarray assay
Total RNA was isolated from cardiomyocytes using the RNeasy Lipit Tissue Mini Kit (Qiagen Inc) and further purified using RNeasy MinElute Cleanup Kit (Qiagen Inc) followed by quality assessment on an Agilent 2100 bioanalyzer. Samples with RIN values 48.0 and a 260/280 ratio between 1.8 and 2.1 were carried forward for cRNA synthesis and hybridization to GeneAtlas MG-430 PM Array Strips (Affymetrix, Santa Clara, CA) following the manufacturer's recommended protocol [3]. Briefly, approximately 250 ng of purified total RNA was reverse transcribed and biotin labeled to produce biotinylated cRNA targets according to the standard Affymetrix GeneAtlas 3 0 -IVT Express labeling protocol (GeneAtlas 3 0 IVT Expression Kit User Manual, P/N 702833 Rev. 4, Affymetrix). Following fragmentation, 6 μg of biotinylated cRNA was hybridized for 16 h at 45°C on the Affymetrix GeneAtlas Mouse MG-430 PM Array Strip. Strips were washed and stained using the GeneAtlas Fluidics Station according to standard Affymetrix operating procedures (GeneAtlas™ System User's Guide, P/N 08-0306 Rev. A January 2010). Strips were subsequently scanned using the GeneAtlas Imager system according to the standard Affymetrix protocol. Fluidics control, scan control, and data collection were performed using the GeneAtlas Instrument Control Software version 1.0.5.267. All microarray analyses were performed by the Wake Forest School of Medicine Microarray Shared Resource Core.

Gene set enrichment analysis (GSEA)
GSEA was performed to determine whether genes belonging to a biological pathway or a previously determined functional group were significantly overrepresented at the top or bottom of a ranked gene list compared to controls without a predefined cut-off value. This bioinformatic tool evaluates all significantly measured targets derived from a microarray experiment at the level of gene sets, which are defined based on prior biological knowledge. Thus, biologically relevant information is not missed by losing target genes due to an "arbitrarily" chosen cut-off value [4]. In this study, expression data of all 21,782 genes were compared against functional gene sets to determine whether any of these sets were enriched in GPER KO cardiomyocytes vs. intact cardiomyocytes.

Acknowledgments
The GPER knockout mouse strain was generated with the help of the KOMP Repository (WWW. KOMP.org) and the Mouse Biology Program (www.mousebiology.org) at the University of California Davis. We appreciate the assistance of Ms. Lou Craddock at Wake Forest University Comprehensive Cancer Center Microarray facility in running the microarray. This work was funded by the National Institutes of Health, USA Grants AG-042758 (L.G.), AG-033727 (L.G.), and HL-051952 (C.M.F.).