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Oxidized Phosphatidylcholines Activate NOX1-Mediated Oxidative Stress Response and Shift Glucose Metabolism in Cardiac Cells

ABSTRACT: In cardiometabolic syndrome the development of cardiovascular disease is linked with an increase in systemic oxidative stress. The formation of free radical species leads to the oxidative modification of lipids, including phosphatidylcholines (OxPCs), which have been implicated in the progression of cardiovascular diseases in humans. We found that reducing plasma levels of OxPCs in mice by AAV-mediated hepatic expression of an OxPC-targeting antibody fragment (scFv-E06), resulted in significant transcriptional changes in the heart, particularly affecting genes involved in metabolism, redox processes, and fibrosis. To investigate the response of cardiac myoblasts to OxPCs in vitro, we exposed H9c2 cells to a defined mixture of OxPC species (OxPAPC). Treatment with OxPAPC resulted in transcriptional upregulation of key metabolic and redox-regulatory pathways, most notably genes regulated by the Nrf2 pathway, including heme oxygenase 1 (Hmox1). OxPAPC induced reactive oxygen species (ROS) production in H9c2 cells through activation NADPH oxidase 1 (Nox1), which upregulated the production of oxidized glutathione. Key metabolic changes after exposure to OxPAPC included a shift towards the pentose phosphate pathway (PPP) and suppression of glycolysis, resulting in overall decreased ATP production. Furthermore, OxPAPC downregulated oxidative phosphorylation in H9c2 cells through a mechanism involving activation of the MEK–ERK mitogen-activated protein kinase (MAPK) pathway. Together, these data demonstrate that in vitro cardiac myoblasts respond to OxPCs by upregulating redox regulatory pathways and shifts in cellular energy production. Furthermore, we identify NOX1 as a novel mediator of OxPC-induced redox stress that may induce cardiac cell damage in cardiometabolic syndrome.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE303370 | GEO | 2025/08/17

REPOSITORIES: GEO

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Project description:Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Here, we show that production of cardiomyocyte NADPH (nicotinamide adenine dinucleotide phosphate), a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with cardiac cell death, fibrosis, and cardiomyopathy. We report that the pentose phosphate pathway (PPP) and mitochondrial serine/glycine/folate metabolic signaling, two major NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced in the heart by pressure overload. We identify ATF4 (activating transcriptional factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these two pathways. Consistent with this, joint pathway analysis (JPA) of transcriptomic and metabolomic data reveals that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in cardiomyocytes in culture leads to a significant increase in NADPH-producing enzymes whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression in vitro strongly augments metabolic flux within these two pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of pressure overload and accelerates the development of heart failure, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Taken together, our findings reveal that ATF4 plays a critical role in cardiac homeostasis under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Project description:NOX1 gene is member of the NADPH-dependent superoxide producing enzyme family. It generates reactive oxygen species (ROS) in regulated manner in response to growth factors, cytokines and calcium signal. Low levels of ROS play role in many processes including cell proliferation, inflammatory response and mitogenesis. NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequence carrying U6 promoter/shRNA cassettes were cloned into GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. Clone 6A and 6C showed 70-80% decrease in NOX1 expression. The cells were expanded and analyzed further. We measured decreased ROS production, cell proliferation and G1/S block of the cell cycle. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. Significant decrease was measured in the volume of forming tumors. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer. Experiment Overall Design: We have found that NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequences carrying the U6 promoter/shRNA cassettes were cloned into a GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. A significant decrease was measured in the volume of tumors formed. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer.

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Oxidized Phosphatidylcholines Activate NOX1-Mediated Oxidative Stress Response and Shift Glucose Metabolism in Cardiac Cells

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