The Role of MCPIP1 in Macrophage Polarization and Cardiac Function Post‐Myocardial Infarction

Figure 1. MCPIP1 Is Expressed In Macrophages After Myocardial Infarction and Its Expression Increases Over Time. A) Volcano plot of differential genes, utilizing three datasets from GEO: GSE166780 (comparing peripheral blood monocytes from AMI patients versus healthy individuals), GSE172270 (comparing monocyte‐derived macrophages from AMI patients versus healthy individuals), and GSE268352 (comparing LPS‐treated versus PBS‐treated human monocyte‐derived macrophages), with Zc3h12a (MCPIP1) highlighted in green. B) Venn contains 116 differential genes (DEGs). GO C) and KEGG D) analyses were conducted using DSEGs. E) Utilizing GSE88924 from GEO, a STEM (Short Time‐series Expression Miner) trend analysis was performed on the gene expression profiles of macrophages in the mouse heart, following the temporal sequence of MI1D, MI7D, and MI14D. F) Module 15, which exhibits significant trend differences, contains 528 genes. The green line represents the expression trend of MCPIP1 over time. G) This figure displays a subset of genes within Module 15, with Zc3h12a (MCPIP1) highlighted in yellow.

Figure 2. Post‐Myocardial Infarction, MCPIP1 is Primarily Expressed by Macrophages. A) MCPIP1 protein and mRNA levels at various time points (sham, 1, 3, 7, 14, and 28 days) post‐MI. n = 3. B) IF of heart at various time points (sham, MI3D, and MI7D), DAPI (blue) stains nuclei, MCPIP1 (red), and CD68 (green) stains macrophages. The line graph represents the distribution of red (MCPIP1) and green (CD68) fluorescence within the tissue sections.(C) Genotype verification of myeloid‐specific MCPIP1 knockout mice. D) Protein levels of MCPIP1 in heart and BMDMs from MCPIP1 mko and MCPIP1 fl/fl mice. n = 3. E) Body weight, heart‐to‐body weight ratio, and spleen‐to‐body weight ratio in littermate mice of the same age (10 weeks). n = 5. F) Survival curves of unoperated MCPIP1mko and MCPIP1fl/fl mice. n = 15. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 3. MCPIP1 mko Mice Exhibit Higher Mortality and Poorer Cardiac Function Following MI. A) Survival curves of MCPIP1mko and MCPIP1fl/fl mice after MI. n = 15. B) Causes of death are categorized as cardiac rupture and other causes. C) Representative cardiac ultrasound images at MI28D. D) Echocardiographic measurements of LVEF, FS, and LVEDV in mice. At least 5 mice per group. E) MASSON and HE staining of hearts from MCPIP1mko and MCPIP1fl/fl mice at different time points (3, 7, 14, and 28 days) post‐MI. F) Cardiac collagen content, infarction angle, and left ventricular thickness in MCPIP1mko and MCPIP1fl/fl mice at MI28D based on MASSON staining results. n = 5. G) Comparison of wet organ weights (heart, liver and lungs) relative to body weight at various time points. n = 5.ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4. MCPIP1 Deficiency Leads to Excessive Macrophage Accumulation. A,B) IF showing the number of macrophages (CD68+) and mean fluorescence intensity in IBZ at different time points (3, 7, 14 days) post‐MI. DAPI (blue) stains nuclei, and CD68 (green) stains macrophages. n = 5 fields of view. C) Protein levels of CD68 and CCL2 in IBZ of mice hearts after MI. n = 3. D) Phalloidin staining revealed the content of F‐actin in BMDMs from MCPIP1mko and MCPIP1fl/fl mice. DAPI (blue) stains nuclei, and F‐actin (green) stains filamentous actin. n = 5 fields of view. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 5. MCPIP1 Plays A Crucial Role in Regulating M2 Polarization Of Macrophages And Cardiac Inflammation. A) IF reveals the percentage of M1 macrophages in IBZ of the hearts. The percentage of (F4/80 + CD86+)/ F4/80+ was calculated. CD86 serves as an M1‐specific marker, while F4/80 is a macrophage‐specific marker. DAPI (blue), F4/80 (green), CD86 (red). n = 5 per field of view. B) IF shows the percentage of M2 macrophages in IBZ of the hearts. The percentage of (F4/80 + Arg1+)/ F4/80+ was calculated. Arg1 is an M2‐specific marker, and F4/80 is a macrophage‐specific marker. DAPI (blue), F4/80 (green), Arg1 (red). n = 5 per field of view. mRNA was extracted from the heart tissues of mice in the sham group and 7 days post‐MI. qRT‐PCR was performed to detect the relative mRNA levels of CD68C), CD86D), Arg1E), IL‐1βF), IL‐10G), TNF‐a H), VCAM‐1I) and CCL2 J). n = 3. BMDMs from MCPIP1mkoand MCPIP1fl/fl mice were treated with LPS (50 ng mL−1) and IL‐10 (20 ng mL−1) respectively to induce classical M1 and M2 phenotypes. WB and qRT‐PCR showed protein and mRNA of CD86 (K) and Arg1(L). n = 3. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 6. Deficiency of MCPIP1 in Macrophages Suppresses Cardiac Fibroblast Activation. A) IF displays the content of type I collagen in IBZ of MCPIP1mko and MCPIP1fl/fl hearts at different time points post‐MI (3, 7, and 14 days), DAPI (blue), Collagen I (green). n = 5 per field of view. B) IF demonstrates MMT in infarcted hearts, calculated as the percentage of (a‐SMA+ F4/80+)/ F4/80+. DAPI (blue), F4/80 (green), a‐SMA (red). n = 5 per field of view. C) qRT‐PCR was performed to detect the relative mRNA levels of Col1, α‐SMA, TGF‐β, and MMP9. n = 3. D) WB was performed to detect the protein level of TGF‐β in BMDMs from MCPIP1 mko and MCPIP1 fl/fl mice after IL‐10 treatment. n = 3. E) BMDMs from MCPIP1 mko and MCPIP1 fl/fl mice were co‐cultured with PCFs from WT mice, respectively. WB analysis was conducted to assess the protein levels of myofibroblast markers F), TGF‐β/Smad3 signaling pathway G) and apoptosis related proteins (H), n = 3. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 7. Ferroptosis in Macrophages Lacking MCPIP1 is Significantly Enhanced. RNA‐seq analysis was performed on BMDMs from MCPIP1 mko and MCPIP1 fl/fl mice, and volcano plots A), heatmaps B), and KEGG) enrichment pathway maps C) were generated based on the differential gene expression. GESA was conducted on DEGs between MCPIP1 fl/fl and MCPIP1 mko BMDMs in the ferroptosis signaling pathway. The GESA plot D) and grouped clustering heatmap E) presented the results. F) Genes with significant differential expression in the ferroptosis signaling pathway are depicted, with red (increased), blue (decreased), and green (no significant difference).

Figure 8. Fer‐1 Attenuates Ferroptosis in MCPIP1‐Deficient Macrophages. A) WB showed protein levels of xCT and GPX4 in MCPIP1 fl/fl and MCPIP1 mko BMDMs treated with PBS and LPS. n = 3. B) qRT‐PCR measured mRNA levels of Ptgs2 and Acsl4. n = 3. C) Spectrophotometric analysis determined relative levels of MDA and GSH. n = 5. D) Protein levels of xCT and GPX4 in MCPIP1 mko BMDMs after LPS (50 ng mL−1,24 h), with Fer‐1 (2µm,6 h) treatment. n = 3. E) Protein levels and mRNA levels of M1 and M2 macrophage markers, CD86 and Arg1, respectively, in BMDMs after Fer‐1 treatment. n = 3. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 9. MCPIP1 Regulates Ferroptosis Through P53. A) Z‐dock prediction of the docking model between MCPIP1 and P53, with MCPIP1 in green, P53 in blue, and the binding domain highlighted in a red box. B) WB showed P53 in MCPIP1 fl/fl and MCPIP1 mko BMDMs after treatment with PBS and LPS. n = 3. C) WB analysis of protein levels of xCT and GPX4 in MCPIP1 mko BMDMs after LPS (50 ng mL−1, 24 h) and PFT‐α (10µm,6 h) treatment. n = 3. D) qRT‐PCR measured mRNA levels of Ptgs2 and Acsl4 in MCPIP1 fl/fl and MCPIP1 mko BMDMs under DMSO, Fer‐1 and PFT‐α treatment. n = 3. E) Spectrophotometric analysis was employed to determine the relative levels of MDA and GSH in MCPIP1 fl/fl and MCPIP1 mko BMDMs treated with DMSO, Fer‐1 and PFT‐α. n = 5. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 10. Inhibition of Ferroptosis in Macrophages Promotes Their Maturation and Activates Cardiac Fibroblasts. A) MCPIP1mko BMDMs were pretreated with LPS and then treated with DMSO, Fer‐1, or PFT‐α. The expression level of CD86 was detected by IF. DAPI (blue), CD86 (red). B) MCPIP1 mko BMDMs were pretreated with IL‐10 and then treated with DMSO, Fer‐1, or PFT‐α. The expression level of Arg1 was detected by IF. DAPI (blue), Arg1 (red). C) The expression level of α‐SMA in PCFs cocultured with BMDMs was detected by IF. DAPI (blue), α‐SMA (red). D) Similarly, P‐Smad3 in PCFs was detected. DAPI (blue), β‐actin (red), P‐Smad3 (green). E) Again, NOX4 in cardiac fibroblasts was detected. DAPI (blue), β‐Tubulin (red), NOX4 (green). F) A wound‐healing assay was performed to assess the proliferation and migration capabilities of PCFs. n = 3.ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 11. Hyperactive Hematopoietic Function in MCPIP1 Mko Mice Spleen. A) Necropsy showed spleen size alterations post‐MI. B) Spleen‐to‐body weight ratio at different time points post‐MI (sham, 3, 7, 14, 28 days). C) HE staining revealed histological changes in splenic architecture post‐MI. D) GSEA enrichment analysis of hematopoietic cell lineage‐associated genes using DEGs in BMDMs from MCPIP1 fl/fl and MCPIP1 mko mice. E) WB analysis of Ki67 protein levels in the spleen post‐MI. F) Complete blood cell counts in MCPIP1 fl/fl and MCPIP1 mko mice pre‐ and post‐MI. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 12. Splenectomy Exerts Protective Effects on Myocardial Infarction in MCPIP1 mko Mice. A) Survival curve analysis of MCPIP1 fl/fl mice. B) Survival curve analysis of MCPIP1 mko mice. C) Effects of splenectomy on the incidence of cardiac rupture in MCPIP1 mko mice. D) Echocardiographic images of MCPIP1 mko mice hearts 28 days post‐splenectomy combined with MI. E) The impact of splenectomy on cardiac function in MCPIP1 mko mice. Each group consists of at least 3 mice. F) Masson's trichrome staining of hearts from MCPIP1 mko mice 28 days post‐splenectomy combined with MI, to detect collagen content, infarction angle, and left ventricular thickness H). n = 5. I) Complete blood cell counts in MCPIP1 mko mice NSE and SE. J) The percentage of CD68+ cells/ total cell number (counted by nuclei) was calculated. DAPI (blue), CD68 (red), n = 5 per field of view. K) The influence of splenectomy on heart‐to‐body weight ratio, liver‐to‐body weight ratio, and lung‐to‐body weight ratio in MCPIP1 mko mice. Each group comprises at least 3 mice. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.