Macrophage PARP7 Alleviates Septic Cardiomyopathy by Interacting With TBK1 and Suppressing TBK1‐Driven Inflammatory Response

Figures

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  FIGURE 1. Screening of the PARP family revealed that PARP7 may be involved in septic cardiomyopathy. (A,B) The heatmap illustrates the expression profiles of PARP family genes in myocardial tissues from mouse and rat models of LPS‐induced sepsis. The RNA‐seq data presented were obtained from the GEO database (mouse: GSE267388, A; rat: GSE266124, B). (C) RT‐qPCR was used to measure the mRNA levels of PARP family members in mouse myocardial tissues following LPS treatment (10 mg/kg, 6 h, n = 8). (D) Integrated RNA‐seq and RT‐qPCR analyses identified PARP7 as a significantly upregulated regulator in LPS‐induced septic cardiomyopathy. (E) Western blot analysis confirmed elevated PARP7 protein levels in myocardial tissues of LPS‐treated mice (n = 6). Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001.

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  FIGURE 2. PARP7 deficiency exacerbates LPS‐induced cardiac dysfunction, histopathological damage, and inflammatory responses in mice. (A) Experimental workflow for WT or Parp7 −/− mice administered normal saline (NS) or LPS (10 mg/kg, 6 h). Cardiac function was assessed 6 h after LPS administration. (B,C) Serum levels of creatine kinase‐MB (CK‐MB, B) and cardiac troponin T (cTnT, C) in WT and Parp7 −/− mice following LPS treatment (n = 7). (D) Representative M‐mode echocardiographic images of the left ventricle. (E,F) Echocardiographic assessment of ejection fraction (EF%, E) and fractional shortening (FS%, F) in mice (n = 7). (G) Representative hematoxylin‐eosin (H&E) staining images. (H–K) Representative immunohistochemical staining images for F4/80 (H) and CD11b (J), along with corresponding quantitative results (I, K) (n = 7). (L–O) The mRNA expression levels of Ifn‐β (L), Tnf‐α (M), Il‐6 (N), and Il‐1β (O) in myocardial tissues from WT and Parp7 −/− mice treated with LPS (n = 7). Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001.

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  FIGURE 3. Single‐nucleus and single‐cell RNA sequencing analyses reveal that PARP7 is predominantly upregulated in macrophages during septic cardiomyopathy. (A,B) Single‐nucleus RNA sequencing (snRNA‐seq) was performed on hearts from LPS‐treated mice (each sample pooled from single‐nuclei suspensions of 3–4 hearts): (A) t‐SNE visualization identified 8 distinct cell populations—smooth muscle cells (SMCs), pericytes (PCs), NK/T cells, neuronal cells, macrophages (Macs), fibroblasts (FBs), endothelial cells (ECs), and cardiomyocytes (CMs); (B) Dot plot illustrating Parp7 expression across these cell types. (C,D) Single‐cell RNA sequencing (scRNA‐seq) was conducted on myocardial tissues from LPS‐treated mice (each sample generated by pooling single‐cell suspensions from 3 to 4 hearts): (C) t‐SNE plot revealing seven major cell types, including NK/T cells, B cells, SMCs, PCs, Macs, FBs, and ECs; (D) Dot plot displaying Parp7 expression patterns within these populations. (E,F) snRNA‐seq was performed on cardiac tissues from patients with heart failure (HF): (E) t‐SNE visualization identified 6 distinct cell populations—SMCs, PCs, Macs, FBs, ECs, and CMs; (F) dot plot illustrating PARP7…

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  FIGURE 4. Macrophage‐specific knockdown of PARP7 aggravates LPS‐induced septic cardiomyopathy in mice. (A) Schematic representation of the experimental design: Mice were administered AAV9‐F4/80‐vector or AAV9‐F4/80‐shPARP7 via tail vein injection at a total dose of 2 × 1011 viral genomes (vg). Four weeks post‐injection, mice were intraperitoneally injected with either normal saline (NS) or lipopolysaccharide (LPS, 10 mg/kg). Cardiac function was assessed 6 h after LPS administration. (B,C) Serum levels of creatine kinase‐MB (CK‐MB, B) and cardiac troponin T (cTnT, C) in mice injected with AAV9‐F4/80‐vector or AAV9‐F4/80‐ shPARP7 and challenged with LPS (n = 7). (D) Representative M‐mode echocardiographic tracings of the left ventricle. (E,F) Echocardiographic assessment of ejection fraction (EF%, E) and fractional shortening (FS%, F) (n = 7). (G) Representative histological images from H&E staining. (H–K) Representative immunohistochemical staining images for F4/80 (H) and CD11b (J), along with quantitative analyses (I, K) (n = 7). (L–O) The mRNA expression levels of Ifn‐β (L), Tnf‐α (M), Il‐6 (N), and Il‐1β (O) in myocardial tissues of mice injected with AAV9‐F4/80‐vector or…

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  FIGURE 5. Macrophage‐specific overexpression of PARP7 attenuates LPS‐induced cardiac dysfunction and inflammatory responses in mice. (A) Schematic representation of the experimental design: Mice were administered AAV9‐F4/80‐vector or AAV9‐F4/80‐Parp7 via tail vein injection at a total dose of 2 × 1011 viral genomes (vg). Four weeks post‐injection, mice were intraperitoneally injected with either normal saline (NS) or lipopolysaccharide (LPS, 10 mg/kg). Cardiac function was assessed 6 h after LPS administration. (B,C) Serum levels of creatine kinase‐MB (CK‐MB, B) and cardiac troponin T (cTnT, C) in mice injected with AAV9‐F4/80‐vector or AAV9‐F4/80‐Parp7 and challenged with LPS (n = 7). (D) Representative M‐mode echocardiographic tracings of the left ventricle. (E,F) Echocardiographic assessment of ejection fraction (EF%, E) and fractional shortening (FS%, F) (n = 7). (G) Representative histological images from H&E staining. (H–K) Representative immunohistochemical staining images for F4/80 (H) and CD11b (J), along with quantitative analyses (I, K) (n = 7). (L–O) The mRNA expression levels of Ifn‐β (L), Tnf‐α (M), Il‐6 (N), and Il‐1β (O) in myocardial tissues of mice injected with…

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  FIGURE 6. Single‐nucleus RNA sequencing reveals that macrophage PARP7 suppresses inflammatory responses during septic cardiomyopathy. (A) Single‐nucleus RNA sequencing (snRNA‐seq) was performed on cardiac tissues from wild‐type (WT) or Parp7 −/− mice treated with LPS (10 mg/kg, 6 h); each sample was pooled from single‐nuclei suspensions of 3–4 hearts. The t‐SNE plot displays the major cell populations, including cardiomyocytes (CMs), endothelial cells (ECs), fibroblasts (FBs), macrophages (Macs), pericytes (PCs), and smooth muscle cells (SMCs). (B) Gene Ontology (GO) enrichment analysis of macrophages (Macs) in cardiac tissues from WT or Parp7 −/− mice following LPS challenge. (C) Heatmaps showing the differential expression gene (DEG) analysis of genes associated with signaling pathways identified by GO enrichment analysis. (D) Network diagram illustrating the potential interactions between macrophages and other cardiac cell types mediated by PARP7, based on CellChat analysis of snRNA‐seq data (the numerical values on the connection lines represent interaction strength). (E) Heatmap showing supernatant cytokine levels detected by mouse cytokine antibody array analysis. Bone marrow‐derived…

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  FIGURE 7. PARP7 interacts with TBK1 to mediate its ADP‐ribosylation, thereby suppressing TBK1‐driven inflammatory response in macrophages during septic cardiomyopathy. (A) Representative images from proximity ligation assay (PLA) showing endogenous interaction between PARP7 and TBK1 in BMDMs treated with LPS (10 µg/mL, 24 h), along with quantitative analysis of red fluorescent signals per cell (n = 6). (B,C) Co‐immunoprecipitation (Co‐IP) analysis of PARP7 and TBK1 in LPS‐treated BMDMs (B) and myocardial tissues from septic mice (C). Endogenous PARP7 was immunoprecipitated using an anti‐PARP7 antibody. (D) Co‐IP of Flag‐PARP7 and His‐TBK1 in NIH/3T3 cells co‐transfected with Flag‐PARP7 and His‐TBK1. Exogenous Flag‐PARP7 was pulled down with an anti‐Flag antibody. (E) Schematic representation of full‐length PARP7 (wild‐type, WT) and its deletion mutants: ∆CCCH (lacking the CCCH domain), ∆WWE (lacking the WWE domain), and ∆PARP (lacking the PARP domain). The catalytic residue histidine 532 (H532) within the PARP domain confers mono‐ADP‐ribosyltransferase activity. (F) NIH/3T3 cells were co‐transfected with His‐TBK1 and either Flag‐PARP7‐WT, ∆CCCH, ∆WWE, or ∆PARP mutants. Cell lysates were…