A Mechanism Study Of TFAM Regulating Cardiomyocyte Pyroptosis Induced By Ionizing Radiation

BackgroundRadiation-induced myocardial injury is a kind of complication with high incidence and fatality rate in radiation-induced heart disease.Previously,it was thought that the onset of symptoms was usually more than 10 years after the end of radiotherapy.However,recent studies have shown that in patients with Non-small cell lung cancer(NSCLC)or esophageal cancer,most cardiac events,including congestive heart failure due to myocardial damage,occurred within the first three years after the end of radiotherapy.As the prognosis of patients with thoracic tumors improves,more radiation-induced myocardial injury will be detected.Therefore,it is urgent for us to deeply explore the mechanism of radiation-induced myocardial injury,find the key molecules that regulate its occurrence,and provide new intervention targets for the prevention and treatment of it.The molecular mechanism of radiation-induced myocardial injury is not fully understood.Currently,it is believed that myocardial injury is mainly related to inflammation,mitochondrial dysfunction,oxidative stress and endoplasmic reticulum stress.Early myocardial injury caused by radiation was mainly caused by inflammation,mitochondrial dysfunction and oxidative stress,and sustained inflammation induced late fibrosis,leading to ventricular remodeling.Previous studies have found that the acute myocardial inflammatory response after radiation may be related to NF-κB signaling pathways,but the specific regulatory mechanism remains unclear.Based on this,a variety of experimental techniques were applied in this study to explore the detailed mechanism of inflammatory response in acute phase of radiationinduced myocardial injury.Firstly,luminex multifactor detection was performed by collecting peripheral blood serum of patients with esophageal cancer before and after radiotherapy.We found that interleukin-1β(IL-1β)and IL-18 increased significantly at the end of radiotherapy and were consistent with the variation trend of cardiac troponin I(cTnI),suggesting that they may play a role in the development of acute radiation-induced myocardial injury.IL-1β and IL-18 are key inflammatory factors in the pyroptosis pathway.Therefore,in the second part,we discussed the mechanism of pyroptosis in acute radiation-induced myocardial injury,providing important theoretical basis for the early prevention and treatment of it.Part Ⅰ:To explore the key inflammatory factors in the process of acute radiationinduced myocardial injury by collecting peripheral blood serum of patients with esophageal cancer before and after radiotherapyObjectives1.To investigate the variation trend of serum cTnI in patients with advanced Esophageal Squamous Cell Carcinoma(ESCC)after Radiation therapy(RT)at different time points.2.The consistency of the change rate of cTnI and inflammatory cytokines in serum before and after RT was analyzed.Methods1.cTnI detection was performed on the sera of 56 patients with advanced ESCC before treatment and at the end of radiotherapy,2 weeks,4 weeks and 8 weeks after radiotherapy,to find the best time point for the observation of acute radiation myocardial injury.2.Serum samples of 15 ESCC patients before treatment and at the end of radiotherapy were collected for Luminex multifactor detection and cTnI detection to explore the key inflammatory factors in the process of acute radiation-induced myocardial injury.Results1.Compared with baseline,cTnI expression increased to the highest level at the end of ESCC radiotherapy and then decreased gradually at 2,4 and 8 weeks.Therefore,we believe that the end of radiotherapy is the best time to observe acute myocardial injury.2.Luminex multifactor detection showed that,compared with baseline,IL-1β,tumor necrosis factor-α(TNF-α),IL-18,IL-6,IL-8,and IL-12 related to inflammation significantly increasesd after radiotherapy.cTnI was significantly elevated at the end of radiotherapy,indicating the occurrence of acute radiation-indiced myocardial mjury.3.Correlation analysis showed that serum ΔcTnl(Δ=(post-radiotherapy level-preradiotherapy level)/pre-radiotherapy level)at the end of radiotherapy was strongly correlated with ΔIL-1β,ΔTNF-α,ΔIL-18,ΔIL-6,ΔIL-8 and ΔIL-12,and the strongest correlation was found with ΔIL-1β and ΔIL-18.Conclusions1.At the end of radiotherapy for esophageal cancer,serum levels of inflammatory factors increased significantly,suggesting that the inflammatory response may be activated.2.The correlation between ΔcTnI and ΔIL-1β and ΔIL-18 was the strongest at the end of radiotherapy,suggesting that IL-1β and IL-18 may play a role in the process of acute radiation-induced myocardial injury.Part Ⅱ:Study on the mechanism of TFAM regulating cardiomyocytes pyroptosis induced by ionizing radiationObjectives1.To construct a mouse model of radiation-induced myocardial injury.2.In vitro and in vivo experiments to verify the role of pyroptosis in acute radiationinduced myocardial injury.3.To explore the molecular mechanism of TFAM regulating cardiomyocyte pyroptosis caused by Ionizing radiation(IR).Methods1.Precise irradiation of mouse whole heart using the Small animal radiotherapy research platform(SARRP)according to dose gradients of 10Gy,20Gy and 30Gy.Hematoxylin-eosin(HE)staining,masson staining,small animal magnetic resonance imaging(MRI)and myocardial injury marker detection were used to observe the changes in cardiac structure and function at 1 day,1 week,4 weeks,12 weeks and 24 weeks after radiation.2.RNA-sequencing(RNA-seq)in the myocardium of IR group and Negtive control(NC)group at 1 and 24 weeks post-radiation was performed respectively,to observe enrichment of differential genes in inflammatory pathways;western blot assay was used to verify the up-regulation of pyroptosis pathway in myocardial tissue and cardiomyocytes after IR treatment in vivo and in vitro.Gsdmd-/-mice and AC 16 and H9C2 GSDMD knockout and overexpressed cell lines were constructed,and neonatal mouse ventricular myocytes(NMVMs)were separated from neonatal mice.western blot,HE and masson staining,MRI and CCK-8 experiments were performed to verify the regulatory role of pyrodeath pathway in radiation-induced myocardial injury.3.Western blot and CCK-8 assay were used to verify the activation of NF-κB pathway after IR to induce pyrodeath of cardiomyocytes in vitro.Precipitation of NFκB p65 in the NLRP3 promoter region was further verified by Chromatin Immunoprecipitation(ChIP)and dual luciferase reporter assay.4.Adeno-associated virus 9(AAV9)vector and TFAM overexpression plasmid were used to construct TFAM overexpression mouse model and AC 16 and H9C2 overexpression cell lines,respectively.The key role of TFAM in myocardial injury after IR was verified in vitro and in vivo by immunofluorescence,western blot,HE and masson staining,MRI and CCK-8.Immunofluorescence,western blot and cytoplasmic DNA PCR amplification were used to explore the molecular mechanism of TFAM regulating cardiomyocytes pyroptosis induced by IR.5.The serum of 56 patients with esophageal cancer included in Part Ⅰ was used to analyze the correlation between the change rate of NF-κB and inflammatory factors in pyroptosis pathway and cTnI and mean heart dose(MHD)before and after radiotherapy.Results1.The mouse model of radiation-induced myocardial injury was establishedHE and masson staining and MRI showed no significant changes in myocardial structure on day 1 after IR.At 1 week after IR,the myocardial structure and function in IR group were significantly changed,including irregular arrangement of myocardial fibers,hyperemia and edema of myocardial cells,infiltration of neutrophils and other inflammatory cells,and significantly decreased ventricular systolic function.At 4,12 and 24 weeks after radiation,the arrangement of myocardial fibers in IR group continued to be disordered,but the acute inflammatory response gradually subsided,lymphocytes,mononuclear macrophages and other cells related to chronic inflammatory response gradually increased,myocardial fibers gradually thickened,and these changes were most obvious at 24 weeks.In addition,the structure and function changes of 20Gy and 30Gy groups are more significant than those of 10Gy groups,and there is no significant difference between 20Gy and 30Gy.Finally,the radiation dose of 20Gy was selected,and the changes of myocardial injury in the acute phase and chronic phase were observed at two time point:1 week and 24 weeks after radiation.2.In vitro and in vivo experiments demonstrated that pyroptosis pathway was up-regulated after IR to myocardium.KEGG pathway analysis showed that NOD-like receptor signaling pathway and NFκB pathway related to inflammation were significantly up-regulated in IR group compared with NC group at 1 week after radiation.GO functional enrichment analysis showed that the pyroptosis pathway was significantly up-regulated in IR group at 1 week after radiation.Western blot showed that the expression of key proteins of pyroptosis pathway was significantly increased in IR group at 1 week after radiation,but there was no significant difference in the expression of pyroptosis pathway between IR group and NC group at 12 weeks and 24 weeks after radiation.In vitro experiments showed that the expression of pyroptosis pathway was upregulated with the increase of radiation dose,and the cell survival rate was decreased with the increase of radiation dose.We finally selected 12Gy,72 hours to carry out the subsequent in vitro experiment.3.Knockout or overexpression of GSDMD proved that pyroptosis was involved in the regulation of radiation-induced myocardial injury.We constructed Gsdmd-/-knockout mice,and western blot showed that the expression level of pyroptosis pathway in IR+Gsdmd-/-group was significantly decreased compared with IR+WT group at 1 week after IR.HE staining and MRI showed that in IR+Gsdmd-/-group,myocardial hyperemia and edema,inflammatory cell infiltration and other changes were significantly reduced,and LVEF and FS were significantly increased.Myocardial fibrosis and ventricular systolic function were also significantly improved in the IR+Gsdmd-/-group at 24 weeks post-radiation.In addition,by constructing AC 16 and H9C2 GSDMD knockout and overexpression cell lines,and separating WT and Gsdmd-/-NMVMs,it was found that IR induced myocardial cell damage was significantly reduced after inhibition of pyroptosis pathway,and significantly increased after activation of pyrodeath pathway,which confirmed the regulatory role of pyroptosis in IR-induced myocardial damage.4.Regulation of radiation-induced cardiomyocytes pyroptosis by NF-κB pathwayWestern blot analysis of cytoplasmic protein and nuclear protein showed that phosphorylated NF-κB p65 increased into the nucleus after myocardial irradiation,indicating the activation of the NF-κB pathway.Before IR,LPS and JSH-23 the activator and inhibitor of NF-κB pathway,were added into AC 16 cell medium,respectively.Western blot and CCK-8 experiments showed that compared with IR group,the expression of the pyroptosis pathway in IR+LPS group and IR+JSH-23 group was significantly up-regulated and down-regulated,respectively,and cell survival rate was significantly decreased and increased,respectively.Dual luciferase assay and ChIP assay showed that NF-κB p65 could directly activate NLRP3 transcription by binding to sites from-1883bp to-1874bp and from-1147bp to1138bp in the upstream promoter region of NLRP3,thereby regulating pyroptosis.5.TFAM regulates myocardial pyrodeath induced by IR through mtDNA/TLR9/NF-κB pathway.First,TFAM overexpression in mice was verified by western blot and immunofluorescence,and then western blot showed that compared with AAV9NC+IR group,phosphorylated NF-κB p65 into the nucleus was significantly reduced in the AAV9-Tfam+IR group,and the expression of pyroptosis pathway was significantly down-regulated,suggesting that TFAM regulates radiation-induced cardiomyocytes pyroptosis by activating the NF-κB pathway.Immunofluorescence co-localization and RT-qPCR showed that dsDNA and mitochondrial co-localization levels decreased in myocardial tissue after IR,while mtDNA significantly increased in cytoplasm.Overexpression of TFAM significantly enhanced dsDNA and mitochondrial co-localization levels and reduced mtDNA cytoplasmic release caused by IR.Overexpression of TFAM can also significantly reduce the production of reactive oxygen species and significantly increase the production of ATP.In vitro experiments,TFAM overexpression plasmid was used to transfect AC 16 and H9C2 cells.Western blot showed that compared with pc-NC+IR group,TFAM expression in pc-TFAM/pc-Tfam+IR group was significantly up-regulated,phosphorylated NFκB p65 into the nucleus was significantly reduced,and the expression of key proteins of pyroptosis pathway is down-regulated,which indicated that TFAM regulates pyroptosis induced by IR through activation of NF-κB pathway.Immunofluorescence,western blot and RT-qPCR showed that down-regulation of TFAM induced the release of mtDNA into cytoplasm,and mtDNA combined with TLR9 to activate the NF-κB pathway,thus inducing pyroptosis of myocardial cells.In addition,immunofluorescence and RT-qPCR showed that the mitochondrial permeability transition pore(mPTP)and Voltage Dependent Anion Channel Protein 1(VDAC1)oligomerization were involved in the release of mtDNA into the cytosol after IR.6.Changes in key inflammatory factors of NF-κB and pyroptosis pathway before and after radiotherapy were investigated in ESCC patientsWe found that the expressions of IL-1β,IL-18 and NF-κB p65 were significantly increased at the end of radiotherapy,and gradually decreased at 2,4 and 8 weeks after radiotherapy.Correlation analysis showed that ΔIL-1β,ΔIL-18 amd ΔNF-κB p65 were significantly and most strongly correlated with ΔcTnI and MHD at the end of radiotherapy,while the correlation decreased at 2,4 and 8 weeks after radiotherapy.Conclusions1.The acute inflammation was the most significant at 1 week after myocardium IR,and the chronic fibrosis was the most significant at 24 weeks after myocardium IR.2.Pyroptosis plays an important regulatory role in acute radiation-induced myocardial injury.3.The downregulation of TFAM induces the release of mtDNA into cytoplasm,binds TLR9 to activate the NF-κB pathway,and then leads to pyroptosis of cardiomyocytes.4.NF-κB p65 activates NLRP3 transcription by binding to the its promoter region.5.Both mPTP and VDAC1 oligomerization are involved in the release of mtDNA into the cytoplasm.6.At the end of radiotherapy,NF-κB p65 and key inflammatory factors in pyroptosis pathway were activated,which may be related to acute radiation-induced myocardial injury.