Abstract

   Hypothermia is widely used in neurosurgery and cardiac surgeries.
   However, little is known about the underlying molecular mechanisms. We
   previously reported that the transcriptome responses of cardiomyocyte
   exposed to hypothermia, “The transcriptome responses of cardiomyocyte
   exposed to hypothermia” [33][4]. Herein, we provide the hypothermia
   inhibited proliferation of cardiomyocyte cells in vitro and the details
   of transcription factors in regulation of differentially expressed
   genes.

   Keywords: Hypothermia, Cardiomyocyte, Transcriptome
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   Specifications Table
   Subject area Biology
   More specific subject area Hypothermia and cardiology
   Type of data Tables and figures
   How data was acquired Polymerase Chain Reaction (Applied Biosystems PCR
   System 7900); Affymetrix GeneChip HTA 2.0 arrays (Affymetrix, Santa
   Clara, USA) were hybridized with biotin-labeled RNA probes.
   Data format Analyzed
   Experimental factors Adult ventricular cardiomyocyte cells (AC16) was
   treated with hypothermia
   Experimental features Cells were cultured at 37 °C or 28 °C with 5%
   CO[2] for 6 h
   Data source location Shenyang city, Liaoning province, China
   Data accessibility Data are presented in this article
   [34]Open in a new tab

   Value of the data
     * •
       The data provides the inhibition of hypothermia on the
       cardiomyocytes in vitro culture.
     * •
       This data provides the details of differentially expressed genes
       (DEGs) of cardiomyocytes exposed on hypothermia.
     * •
       The data may stimulate further research on the function of
       transcription factor (TF) stimulated in cardiomyocytes under
       hypothermia.

1. Data

   The viable cell number was determined by Cell Counting Kit-8 (CCK-8)
   assay ([35]Fig. 1). As shown in [36]Fig. 1, the relative cell number of
   hypothermia was decreased as the time of hypothermia culture.

Fig. 1.

   [37]Fig. 1.
   [38]Open in a new tab

   Hypothermia inhibited proliferation of cardiomyocyte cells.

   Cell proliferation was detected by CCK-8 assay at various time points
   according to the guidance of the manufacturer.

   The details of changed genes are listed in Supplementary [39]Table S1.

   Pathway enrichment analysis was performed considering the notion that
   different genes cooperate with each other to exercise their biological
   functions. The changed pathways are listed in Supplementary [40]Table
   S2.

   The details of TFs in regulation of DEGs are listed in Supplementary
   [41]Table S3.

   Primers for the 11 randomly selected differentially expressed genes are
   listed in Supplementary [42]Table S4. The amount of 18s, a constitutive
   transcript (endogenous control) was normalized to check the fold change
   in the expression of the target genes ([43]Fig. 2).

Fig. 2.

   [44]Fig. 2.
   [45]Open in a new tab

   qRT-PCR validation of 11 differentially expressed genes during
   hypothermia stress. Dickkopf 1 homolog (DKK1), SMAD family member 7
   (SMAD7), Rho-related BTB domain containing 3 (RHOBTB3), Kruppel-like
   factor 5 (KLF5), Suppressor of cytokine signaling 4 (SOCS4),
   Cyclin-dependent kinase 1 (CDK1), Suppressor of defective silencing 3
   homolog(SUDS3), Zinc finger protein 17 (ZNF17), Mediator complex
   subunit 23 (MED23), Zinc finger, DHHC-type containing 17 (ZDHHC17), Son
   of sevenless homolog 2 (SOS2).

2. Experimental design, materials and methods

2.1. Experimental design and hypothermia treatment

   AC16 human adult ventricular cardiomyocytes were cultured in incubator
   with normal temperature (37 °C) and 5% CO[2]. The hypothermia treated
   AC16 cells were cultured in another incubator with low temperature
   (28 °C) and 5% CO[2]. The cells incubated for six hours and the RNA was
   extracted using the TRIzol (Invitrogen) reagent. This experiment was
   repeated three times (N=3). Then, the RNA was isolated by Genminix Co.
   (Shanghai, China). Finally, the microarray hybridization was completed
   [46][4].

2.2. Cells culture

   AC16 human adult ventricular cardiomyocytes were purchased from the
   American Type Cell Culture (ATCC) [47][2]. The cells were maintained in
   DMEM/F-12 supplemented with 10% fetal bovine serum, 100 units/ml
   penicillin, and 100 μg/ml streptomycin. Cells were cultured at 37 °C or
   28 °C with 5% CO[2].

2.3. Cell Counting Kit-8 (CCK8) assay

   Cell viability was determined by the Cell Counting Kit-8 assay
   (DOJINDO, Japan), according to the manufacturer׳s instructions. The
   absorbance of each well was measured at 450 nm with a microtiter plate
   reader.

2.4. Microarray hybridization for the hypothermia regulated transcriptome

   Microarray hybridization has been described previously in [48][4].

2.5. GO analysis

   Gene Ontology (GO) analysis was applied to analyze the main function of
   the differentially expressed genes according to the gene ontology,
   which is the key functional classification of NCBI that can organize
   genes into hierarchical categories and uncover the gene regulatory
   network based on biological process and molecular function [49][1].

3. Pathway analysis

   Pathway analysis was used to identify the significant pathway of the
   differential genes according to KEGG, Biocarta and Reactome. Fisher׳s
   exact test was used to select the significant pathway, and the
   threshold of significance was defined by P-value and FDR. The
   enrichment Re was calculated based on the previously published equation
   [50][3].

4. Statistical analysis

   RVM t-test was applied to filter the differentially expressed genes for
   the control and hypothermia treated group because the RVM t-test can
   raise degrees of freedom effectively in the cases of small samples.
   After the significant analysis and FDR analysis, we selected the
   differentially expressed genes according to the p-value threshold. P
   value <0.05 was considered as significant difference [51][4].

Acknowledgments