TFAM Modulates Cardiomyocytes Pyroptosis Induced by Ionizing Radiation through mtDNA/TLR9/NF-kB Pathway

Purpose/Objective(s)Mitochondrial transcription factor A (TFAM) is a pivotal factor for regulating mitochondrial DNA (mtDNA) replication, transcription and biogenesis. Previous studies have reported that cytosolic mtDNA stress can lead to cardiomyocytes pyroptosis, which is characterized by inflammasome formation. In this study, we attempted to investigate the mechanism of TFAM regulate cardiomyocytes pyroptosis induced by ionizing radiation.Materials/MethodsThe peripheral blood serum of patients with esophageal cancer before and after definitive chemoradiotherapy was collected for Luminex multiplex cytokine assays. C57BL/6 mice were irradiated with the whole heart using small animal radiation research platform (SARRP) to construct a radiation-induced myocardial damage (RIMD) mouse model, and the ventricular function was evaluated using 9.4T Bruker magnetic resonance (MR) scanner. The function changes of cardiomyocytes exposed to radiation were observed in vitro and in vivo after knocking out GSDMD. Furthermore, the changes of mitochondrial function, the levels of cytosolic mtDNA, and the protein levels of NF-kB and pyroptosis pathway in irradiated cardiomyocytes were analyzed by knockdown and overexpression of TFAM in vitro and in vivo.ResultsBy multifactor cytokine assays we found that pyroptosis related IL-1β and IL-18 were significantly increased in patients with high mean heart dose (MHD) after radiotherapy, while those with low MHD were not significantly increased after radiotherapy. Next, we successfully constructed the RIMD mouse model using a single heart irradiation of 20 Gy. We found that the gene expression of pyroptosis pathway was significantly up-regulated after cardiac irradiation by myocardial tissue transcriptomic sequencing. Compared with wild-type (WT) mice, cardiac systolic function of Gsdmd−/− mice was significantly improved at 1, 2, 6, 12, and 24 weeks after heart irradiation. In vitro, we also demonstrated increased viability of irradiated cardiomyocytes by knocking out GSDMD. In vitro and in vivo experiments confirmed the expression of TFAM decreased after radiation. By overexpression of TFAM, we found that irradiated cardiomyocytes showed improved mitochondrial function, decreased release of mtDNA into cytoplasm through mitochondrial permeability transition pores (mPTPs), decreased binding of cytosolic mtDNA to TLR9, and decreased expression of NF-kB and pyroptosis pathway proteins. Dual luciferase gene reporter assays and Chromatin immunoprecipitation (CHIP) assay confirmed that p65 could bind the NLRP3 promoter region. In addition, we found that ventricular function deteriorated and improved in mice with knockdown and overexpression of TFAM through adeno-associated virus serotype 9 (AAV9), respectively.ConclusionOur study indicated that TFAM regulate irradiated cardiomyocytes pyroptosis through mtDNA/TLR9/NF-kB pathway. We provide a novel mechanism of RIMD, revealing an underappreciated intervention target for RIMD. Mitochondrial transcription factor A (TFAM) is a pivotal factor for regulating mitochondrial DNA (mtDNA) replication, transcription and biogenesis. Previous studies have reported that cytosolic mtDNA stress can lead to cardiomyocytes pyroptosis, which is characterized by inflammasome formation. In this study, we attempted to investigate the mechanism of TFAM regulate cardiomyocytes pyroptosis induced by ionizing radiation. The peripheral blood serum of patients with esophageal cancer before and after definitive chemoradiotherapy was collected for Luminex multiplex cytokine assays. C57BL/6 mice were irradiated with the whole heart using small animal radiation research platform (SARRP) to construct a radiation-induced myocardial damage (RIMD) mouse model, and the ventricular function was evaluated using 9.4T Bruker magnetic resonance (MR) scanner. The function changes of cardiomyocytes exposed to radiation were observed in vitro and in vivo after knocking out GSDMD. Furthermore, the changes of mitochondrial function, the levels of cytosolic mtDNA, and the protein levels of NF-kB and pyroptosis pathway in irradiated cardiomyocytes were analyzed by knockdown and overexpression of TFAM in vitro and in vivo. By multifactor cytokine assays we found that pyroptosis related IL-1β and IL-18 were significantly increased in patients with high mean heart dose (MHD) after radiotherapy, while those with low MHD were not significantly increased after radiotherapy. Next, we successfully constructed the RIMD mouse model using a single heart irradiation of 20 Gy. We found that the gene expression of pyroptosis pathway was significantly up-regulated after cardiac irradiation by myocardial tissue transcriptomic sequencing. Compared with wild-type (WT) mice, cardiac systolic function of Gsdmd−/− mice was significantly improved at 1, 2, 6, 12, and 24 weeks after heart irradiation. In vitro, we also demonstrated increased viability of irradiated cardiomyocytes by knocking out GSDMD. In vitro and in vivo experiments confirmed the expression of TFAM decreased after radiation. By overexpression of TFAM, we found that irradiated cardiomyocytes showed improved mitochondrial function, decreased release of mtDNA into cytoplasm through mitochondrial permeability transition pores (mPTPs), decreased binding of cytosolic mtDNA to TLR9, and decreased expression of NF-kB and pyroptosis pathway proteins. Dual luciferase gene reporter assays and Chromatin immunoprecipitation (CHIP) assay confirmed that p65 could bind the NLRP3 promoter region. In addition, we found that ventricular function deteriorated and improved in mice with knockdown and overexpression of TFAM through adeno-associated virus serotype 9 (AAV9), respectively. Our study indicated that TFAM regulate irradiated cardiomyocytes pyroptosis through mtDNA/TLR9/NF-kB pathway. We provide a novel mechanism of RIMD, revealing an underappreciated intervention target for RIMD.