Mechanism Of Gasdermin D-Mediated Endotoxin-induced Myocardial Dysfunction

Background:Cardiac dysfunction,an early complication of sepsis and endotoxemia,is the major cause of death in intensive care units.Endotoxin-induced myocardial dysfunction（EIMD）can increase the mortality of endotoxemia patients by about 50–70%.However,there is no specific therapy available for EIMD at present.Aim:To elucidate the key mechanisms of EIMD to develop a novel therapeutic target is to provide the sound theoretical basis for endotoxemia.Methods:1.Phenotypically:to explore the key mechanisms of initiating endotoxin-induced myocardial dysfunction:（1）A model of endotoxin-induced myocardial dysfunction was constructed in 8 weeks old C57BL/6J wild-type（WT）male mice,echocardiography,Mitosox staining,Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick-end labeling（TUNEL）,and transmission electron microscopy（TEM）were used to determine the early stage which was characterized by the onset of left ventricular systolic dysfunction and the later stage which was characterized by the onset of left ventricular（LV）mass increase;（2）In human cardiomyocytes AC-16,the model of endotoxin-induced cell injury was constructed,Mitosox staining,Lactate dehydrogenase release（LDH）assay,and TEM were used to determine the early time point for initiating mitochondrial oxidative stress without cardiomyocytes death and the late time point as the onset of cardiomyocytes apoptosis;（3）Moreover,bulk RNA sequencing（bulk RNAseq）in myocardium was used to investigate the target genes for EIMD.The related gene-knockout model,echocardiography,Mitosox,Western blot,and TEM were used to confirm the target gene is gasdermin D（Gsdmd）.2.Molecular mechanically:mitochondria and cell membrane were isolated from AC16 cells and HEK293T cells,Western blot,Blue-Native PAGE（BN-page）,co-immunoprecipitation,and ultra-High performance liquid chromatography-high-resolution tandem mass spectrometry（UHPLC-HRMS/MS）analysis were used to clarify that the direct target of GSDMD-mediated endotoxin-induced myocardial dysfunction is oxidized cardiolipin and its mechanism.The upstream regulatory factors of mitochondrial oxidized cardiolipin involved in endotoxin-induced myocardial dysfunction were further explored.Results:1.Key mechanisms of initiating endotoxin-induced myocardial dysfunction:（1）In WT mice,a model of endotoxin-induced myocardial dysfunction was constructed;The results revealed that left ventricular systolic dysfunction,mitochondrial oxidative stress dysfunction,and mitochondrial pore formation occurred without cardiomyocytes death after the 4-hour Lipopolysaccharide（LPS）challenge as the early stage,while LV mass increased,cell death and cell membrane pore formation occurred after 14-hour LPS exposure as the late stage;（2）In AC16 cells,a model of endotoxin-induced cell injury was constructed,the results showed that mitochondrial oxidative stress and pores formation initiated without cardiomyocytes death after 2 hours of LPS 10 ng/m L exposure as the early time point,while cell membrane pore formation and cardiomyocytes death began to appear after 10 ng/m L LPS exposure for 4h as the late time point;（3）In vivo and vitro,Mito Tempol,a specific m ROS scavenger,can significantly improve heart function,diminish m ROS level and mitochondrial pores at early LPS challenge,reduce cardiomyocytes apoptosis and cell membrane pores at late LPS exposure;（4）The results of Bulk RNAseq showed that Gsdmd,Nlrp3,Tlr4,Caspase 1,and Caspase 4/11 significantly activated in the myocardium after 4-hour LPS treatment;（5）In vivo and vitro,deficiency of Nlrp3,Caspase 1,Caspase 4/11,and Gsdmd can significantly reduce mitochondrial membrane pore formation and oxidative stress dysfunction,cardiac dysfunction and the cleavage of GSDMD.2.Gasdermin d mediates endotoxin-induced myocardial dysfunction directly via oxidized cardiolipin:（1）In vivo,vitro,and exogenous transfection,the N-terminal gasdermin D（GSDMD-N）was found to aggregate to mitochondria and form oligomers during early endotoxemia.The L192 residue of GSDMD-N probably participates in its localization and oligomerization in mitochondria;（2）The model of endotoxin-induced myocardial dysfunction was established in Gsdmd^fl/fland Gsdmd-CKO mice.The results revealed that Gsdmd-CKO mice significantly reversed LPS-induced cardiac dysfunction,m ROS production,mitochondrial membrane pores,cardiomyocytes apoptosis,and mortality compared with Gsdmd^fl/flmice;（3）The quantity of mitochondrial oxidized cardiolipin（ox CLs）significantly increased in heart tissue after 4 hours of LPS exposure.Further,treatment with XJB-5-131,a specific CL oxidation inhibitor,could inhibit early endotoxin-induced ox CLs accumulation;（4）XJB-5-131 or SS-31,or transfection of si RNA against CL synthase（CLS）or phospholipid scramblase-3（PLS3）can inhibit ox CLs accumulation,aggregation and oligomerization of GSDMD-N in mitochondria after early endotoxemia;（5）XJB-5-131 or SS-31 could inhibit cardiac dysfunction,reduction in m ROS production,mitochondrial membrane pore formation and cardiomyocytes apoptosis,improve the survival rate in mice after LPS challenge;（6）The GSDMD pore is prior to BAX/BAK and VDAC pores in mitochondria in early endotoxemia,and initiate BAX and VDAC1oligomerization in mitochondria at the late stage of endotoxemia.And XJB-5-131 or transfection of PLS3-si RNA can inhibit the oligomerization of mitochondrial BAX and VDAC1 in late endotoxemia.3.The upstream regulator of oxidized cardiolipin to mediate endotoxin-induced myocardial dysfunction:（1）In AC-16 cells,the mitochondrial complex I-V inhibitors were pretreated,respectively,and then primed with Poly（I:C）1μg/m L for 3 hours,followed by LPS 10ng/m L for 2 hours,the results showed that only complex II inhibitors dimethyl itaconate（DI）or dimethyl malonate（DMM）rather than inhibitors of other complexes could significantly inhibit the production of m ROS and the aggregation and oligomerization of GSDMD-N in mitochondria;（2）DI or DMM could significantly improve cardiac function,reduce ox CL levels,m ROS production and mitochondrial membrane pore formation,and cardiomyocytes death during EIMD.Conclusions:The study revealed:（1）Mitochondrial stress dysfunction is the key regulatory mechanism of early endotoxemia-related myocardial dysfunction;（2）GSDMD cleaved by the activated canonical and non-canonical inflammasomes involved in early endotoxemia-associated cardiac dysfunction;（3）GSDMD oligomerization facilitates mitochondrial pore formation and cardiac dysfunction during early endotoxemia,externalizative ox CL is the direct target;（4）The GSDMD pore is earlier than BAX/BAK and VDAC pores in early endotoxin-induced myocardial dysfunction;（5）Mitochondrial complex II is the major contributor to CL oxidation in cardiomyocytes during EIMD.In sum,modulation of cardiolipin oxidation and oxidized cardiolipin–GSDMD interaction could be an early therapeutic direction for EIMD.