System level approaches to identify key regulators of cell death during myocardial infarction

Abstract

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Although myocardial cell death plays a significant role in myocardial infarction (MI), its underlying mechanism remains to be elucidated. The need to conduct genome-scale (omics) experiments has emerged to identifies biological pathways that are enriched in a gene list more than might be expected by chance. In this study, differentially expressed genes, miRNAs and proteins are obtained using next-generation sequencing (NGS) and tandem mass tag (TMT) based mass spectrometry. Integrative analysis of transcriptomic and proteomic analysis identified changes in biological processes during the pathogenesis of MI. Among them the cell death processes are the main processes in MI, I focused on finding key regulators of the cell death using regulation of specific microRNA, mRNA and inhibition of proteins. The first part of this study was a systems biological approach to identify DEGs and DEmiRs in myocardial infarction (MI) using RNA sequencing data in an MI mouse model. The results of the high throughput approach identified a pair of functionally antagonistic miRNAs and their targets in MI. The two miRNAs were miR-30-5p (family) and miR-142a-5p, which were downregulated and upregulated in MI, respectively. Gene Set Enrichment Analysis (GSEA) using the predicted targets of the two miRNAs suggested that apoptosis is an essential gene ontology (GO)-associated term. In vitro functional assays using neonatal rat ventricular myocytes (NRVMs) demonstrated that miR-30-5p is anti-apoptotic and miR-142a-5p is pro-apoptotic. Luciferase assays showed that the apoptotic genes, Picalm and Skil, and the anti-apoptotic genes, Ghr and Kitl, are direct targets of miR-30-5p and miR-142a-5p, respectively. siRNA studies verified the results of the luciferase assays for target validation. The system-level high throughput analysis of two-layer omics data identified a pair of functionally antagonistic miRNAs and their targets in MI. The second part of this study was to examine the pathogenesis of MI, and to identify the potential therapeutic targets using tandem mass tag (TMT)-based quantitative proteomic approach. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) revealed that the glutathione metabolic pathway and reactive oxygen species (ROS) pathway were significantly downregulated during MI. In particular, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis (an iron-dependent program of regulated necrosis), was downregulated in the early and middle stages of MI. RNA-seq and qRT-PCR analyses suggested that GPX4 downregulation occurred at the transcriptional level. Depletion or inhibition of GPX4 using specific siRNA or the chemical inhibitor RSL3, respectively, resulted in the accumulation of lipid peroxide, leading to cell death by ferroptosis in H9c2 cardiomyoblasts. Although neonatal rat ventricular myocytes (NRVMs) were less sensitive to GPX4 inhibition than H9c2 cells, NRVMs rapidly underwent ferroptosis in response to GPX4 inhibition under cysteine deprivation. This study suggests that downregulation of GPX4 during MI contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress such as cysteine deprivation. The third part of this study was to examine the functional role of phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1), the intrinsic inhibitor of PI3K, in light that phosphatidylinositol-3-kinase -AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation and apoptosis. I found that PIK3IP1 expression level was significantly downregulated in an MI mouse model. The MTT assay demonstrated that cell viability decreased significantly with treatment of H2O2 (200 -500 μM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 μM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase–3, were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced, and PI3K-mediated, apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. This results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. These findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interacting with the ETA-PI3Kr-AKT axis. Taken together, my thesis study could provide invaluable in-depth information concerning the pathogenesis of MI which could lead to the development of therapeutic tools. This system-level approaches could also be used to identify pathogenesis of other cardiac diseases.