The Novel Non-coding Transcriptional Regulator Gm18840 Drives Cardiomyocyte Apoptosis in Myocardial Infarction Post Ischemia/Reperfusion

Abstract

Background: Ischemia/reperfusion-mediated myocardial infarction (MIRI) is a major pathological factor implicated in the progression of ischemic heart disease, but the key factors dysregulated during MIRI have not been fully elucidated, especially those essential non-coding factors required for cardiovascular development.

Methods: A murine MIRI model and RNA sequencing (RNA-seq) were used to identify key lncRNAs after myocardial infarction. qRT-PCR was used to validate expression in cardiac muscle tissues and myocardial cells. The role of Gm18840 in HL-1 cell growth was determined by flow cytometry experiments in vitro. Full-length Gm18840 was identified by using a rapid amplification of cDNA ends (RACE) assay. The subcellular distribution of Gm18840 was examined by nuclear/cytoplasmic RNA fractionation and qRT-PCR. RNA pulldown and RNA immunoprecipitation (RIP)-qPCR assays were performed to identify Gm18840-interacting proteins. Chromatin isolation by RNA purification (ChIRP)-seq (chromatin isolation by RNA purification) was used to identify the genome-wide binding of Gm18840 to chromatin. The regulatory activity of Gm18840 in transcriptional regulation was examined by a luciferase reporter assay and qRT-PCR.

Results: Gm18840 was upregulated after myocardial infarction in both in vivo and in vitro MIRI models. Gm18840 was 1,471 nt in length and localized in both the cytoplasm and the nucleus of HL-1 cells. Functional studies showed that the knockdown of Gm18840 promoted the apoptosis of HL-1 cells. Gm18840 directly interacts with histones, including H2B, highlighting a potential function in transcriptional regulation. Further ChIRP-seq and RNA-seq analyses showed that Gm18840 is directly bound to the cis-regulatory regions of genes involved in developmental processes, such as Junb, Rras2, and Bcl3.

Conclusion: Gm18840, a novel transcriptional regulator, promoted the apoptosis of myocardial cells via direct transcriptional regulation of essential genes and might serve as a novel therapeutic target for MIRI.

Keywords: Gm18840; cardiomyocytes apoptosis; lncRNA; myocardial infarction; transcriptional regulation.

FIGURE 1

Identify lncRNAs that are differentially expressed upon myocardial ischemia–reperfusion injury. (A) The workflow of experimental design. Overall view of myocardial ischemia–reperfusion injury-related lncRNA identification and functional and mechanistic analysis of Gm18840 in myocardial ischemia–reperfusion injury. (B) Distinct gene expression patterns in cardiac tissues with or without IRI treatment. PCA plot showing the clustering of the RNA expression profile of cardiac tissue in mice with or without IRI. The red dots represent the IRI samples, and the blue dots represent the normal samples. (C) Heatmap showing differentially expressed genes in cardiac tissues with or without IRI. Detailed differentially expressed genes are provided in Supplementary Tables 1, 2,. (D) Gene ontology analysis of differentially expressed genes in cardiac tissues with or without IRI treatment. (E) Illustration of differentially expressed lncRNAs in cardiac tissues upon IRI treatment. (F) Validation of the differentially expressed lncRNAs by qRT-PCR in cardiac tissues with or without IRI treatment. ***p < 0.001.

FIGURE 2

Validation of identified lncRNAs using a simulated ischemia/reperfusion model in HL-1 cells. (A,B) RNA-seq analysis in an in vitro model of simulated ischemia. HL-1 cells were cultured under normoxia or hypoxia. Differentially expressed genes between normoxia and hypoxia in HL-1 cells are shown (A). Gene ontology analysis was performed using the differentially expressed genes (B). (C) Validation of the differentially expressed lncRNAs by qRT-PCR in the murine cardiac muscle cell line HL-1 upon hypoxia treatment. HL-1 cells were cultured with 20% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 h. The expression of these genes was normalized to GAPDH.

FIGURE 3

Knockdown of Gm18840 reduced hypoxia-induced apoptosis in cardiac muscle cells. (A,B) Knockdown of Gm18840 decreased hypoxia-resultant apoptosis in HL-1 cells. Representative flow cytometry (A) and statistical analysis (B) are shown. HL-1 cells were treated with siRNA targeting Gm18840 before hypoxia experiments. For the hypoxia experiments, HL-1 cells were cultured in 20% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 h. The cells were harvested 24 h after exposure to hypoxia or normoxia culture. (C) Morphology of HL-1 cells under normoxia, hypoxia with negative control (NC) siRNA, or hypoxia with siRNA targeting Gm18840. ***p < 0.001.

FIGURE 4

Identification and characterization of Gm18840. (A) Schematic diagram of Gm18840 from Ensembl RefSeq and RACE. Blue boxes: exons annotated by NCBI RefSeq. Red boxes: additional sequences and exons determined by RACE. Blue lines: introns. Arrows on blue lines indicate transcriptional directions. (B) Gel electrophoresis of nested PCR products from 5′-RACE and 3′-RACE. Red arrows indicate product bands. (C) The full-length sequence of the Gm18840 transcript. Blue: reference sequence from NCBI RefSeq. Red: sequence from RACE. (D,E) ORF finder software (D) and CPAT software. (E) Prediction of the protein-coding potential of Gm18840.

FIGURE 5

Gm18840 interacted with histone H2B and was located in the nucleus and cytoplasm. (A) Schematic illustration of RNA pulldown assays. Biotinylated DNA against the Gm18840 lncRNA was used for RNA pulldown analysis. HL-1 cells were fixed with formaldehyde and sonicated. The Gm18840 lncRNA was hybridized with DNA probes, and the interacting protein of Gm18840 was captured. Primers against Gapdh were used as the negative control. (B) Silver staining showing the proteins uniquely bound by Gm18840 in HL-1 cells. (C) LC-MS/MS analysis of the Gm18840-interacting proteins. (D) RIP-qPCR analysis of the direct binding between H2B protein and the Gm18840 lncRNAs in HL-1 cells. (E) Distribution of Gm18840 in the cytoplasm and the nucleus. RNA was extracted from the cytoplasm and the nucleus, and the expression of U6 (mainly distributed in the nucleus), Gapdh (mainly distributed in the cytoplasm), and Gm18840 was detected. ***p < 0.001.

FIGURE 6

Gm18840 is involved in transcriptional regulation in cardiac muscle cells. (A) Genome-browser screen showing the binding of Gm18840 in the regulatory regions of representative genes. ChIRP-seq experiments were conducted to determine the genome-wide binding of Gm18840 to chromatin. Representative binding among the regulatory regions of Wdr77, Junb, Ly6a, and Bcl3 is shown. (B) Distribution of Gm18840-bound regions. The numbers of Gm18840-bound peaks in the gene body, promoter, and intergenic regions are shown. (C) Motif analysis of Gm18840-bound regions. The motif analysis was performed with the Homer suite. The EWSR1-FLI1, E2F6, ZNF263, ETS, SP1, and MEIS2 motifs were highly enriched, implying the coregulation of Gm18840 with these factors. (D) Gene ontology analysis of genes associated with Gm18840-bound peaks. (E) Gm18840 directly regulated the transcriptional activity of its targets. Luciferase reporter assays were performed in HL-1 cells. The representative regions bound by Gm18840, including Junb, Rras2, and Bcl3, were cloned into the pGL3 plasmid. The luciferase constructs were cotransfected with the pcDNA3.1 vector or pcDNA3.1-Gm18840 plasmids. The firefly luciferase activity was normalized to Renilla luciferase activity. **p < 0.01 and ***p < 0.001.

FIGURE 7

Gm18840 partially restored the transcriptional pattern under hypoxia to that under normoxia. (A) PCA of the transcriptome of HL-1 cells under normoxia and HL-1 cells transfected with negative control siRNA or siRNA targeting Gm18840 under hypoxia. The transcriptomes of HL-1 cells under normoxia and HL-1 cells with Gm18840 knockdown under hypoxia were similar. (B) Knockdown of Gm18840 under hypoxia partially restored gene expression to that under normoxia. Heatmap showing the expression of genes differentially expressed in normoxia (Normal group) compared to hypoxia (NC group). Relative expression of these genes in HL-1 cells under normoxia (Normal group) and HL-1 cells transfected with negative control siRNA or siRNA targeting Gm18840 under hypoxia (SiRNA group) are shown. (C) Gene ontology analysis of genes directly regulated by Gm18840. Genes directly regulated by Gm18840 were defined by ChIRP-seq binding and differentially expressed upon Gm18840 knockdown.