S100A12 triggers NETosis to aggravate myocardial infarction injury via the Annexin A5-calcium axis

Abstract

Neutrophil extracellular traps (NETs) play a critical role in acute myocardial infarction (AMI) and the externalization of S100 family members. Here, we show the effects of S100A12 on NETs formation and myocardial injury following AMI. S100A12 expression increases rapidly in neutrophils and peaks on day 1 after AMI, promoting NETs production and exacerbating myocardial injury. DNase I, an inhibitor of NETs, reduces apoptosis of cardiomyocytes induced by S100A12. Mechanistically, the interaction of S100A12 and Annexin A5 (ANXA5) enhances calcium influx and promotes NETs formation. Blockage of ANXA5 effectively attenuates heart function impairment after AMI. Finally, we show that plasma S100A12 levels correlate with dsDNA concentration, and this correlation is associated with an increased risk of all-cause mortality during the 1-year follow-up of AMI patients. These findings, derived from male mice, reveal the S100A12-ANXA5-calcium influx axis as a potential therapeutic target and biomarker for AMI.

Fig. 1. S100A12 expression aggravated myocardial infarction (MI) injury.

a Representative echocardiographic recordings at Day 28 after MI or sham operation. b–i Echocardiographic parameters were analyzed (n = 8 biological replicates). j Representative Masson and Hematoxylin & eosin and staining of cardiac tissue obtained from WT and TG mice at Day 28 after MI or sham operation. Quantitative analysis of scar circumference (k) and infarct size (l) at Day 28 post-MI in WT and TG mice (n = 4 biological replicates). Data are expressed as mean ± SEM. Statistical analyses were carried out using two-tailed unpaired t-test for (k), (l), one-way ANOVA with Tukey’s multiple comparisons test for (b), (f), (g), Brown-Forsythe and Welch’s ANOVA test for (c), (d), (e), and Kruskal-Wallis test with Dunn’s multiple-comparison test for (h), (i). Source data are provided as a Source Data file.

Fig. 2. Content of S100A12 significantly increased in the myocardial tissue of TG mice during the early stages after MI, in parallel to leukocyte infiltration and neutrophil extracellular traps (NETs) formation.

a, b Representative immunohistochemical (IHC) staining of S100A12 and quantification in the border area of infarcted myocardial tissue of TG mice at different time points after MI (n = 4 biological replicates). Scale bar, 50 μm. c S100A12 mRNA expression level was analyzed in the border area of TG mice at different time points after MI (n = 6 biological replicates). d, e Representative blots of S100A12 and quantitative analysis in the border area of TG mice at different time points after MI (n = 4 biological replicates). f–h Immunofluorescence double staining of neutrophil marker (Ly6G) and S100A12 in the border area of TG and WT mice and quantification at day 1 post-MI (n = 4 biological replicates). Scale bar, 20 μm. i, j Representative blots of NETs-related proteins [myeloperoxidase (MPO), neutrophil elastase (NE), and citrullination of histone 3 (citH3)] and quantification in WT and TG mice at Day 1 after MI or sham operation. One-way ANOVA with Tukey’s multiple comparisons test is used for statistical analyses of MPO, Brown-Forsythe and Welch’s ANOVA test is used for analyses of NE and citH3 (n = 4 biological replicates). k–n Representative IHC staining of NET-related proteins and quantification in WT and TG mice at Day 1 post-MI (n = 4 biological replicates). Scale bar, 50 μm. Data are expressed as mean ± SEM. Statistical analyses were carried out using one-way ANOVA with Tukey’s multiple comparisons test for (h), (l), (m), (n) and Brown-Forsythe and Welch’s ANOVA test for (b), (c), (e), (g). Source data are provided as a Source Data file.

Fig. 3. Expression of S100A12 led to neutrophil extracellular traps (NETs) formation.

Bone marrow-derived neutrophils (BMDNs) were isolated from the WT and TG mice. a SYTOX Green staining was performed to observe the rate of NETosis in BMDNs from TG and WT mice. Scale bar, 10 μm. b Analysis of NETosis rate (n = 4 biological replicates). c, d Immunofluorescence double staining and quantification of myeloperoxidase (MPO) and citrullination of histone 3 (citH3) in BMDNs from TG and WT mice. Scale bar, 50 μm. (n = 4 biological replicates). e, f Representative blots of NETs-related proteins [MPO, neutrophil elastase (NE), citH3, and peptidylarginine deiminase 4 (PAD4)] and quantification of TG and WT primary neutrophils. Two-tailed unpaired t-test is used for statistical analyses of MPO and PAD4, unpaired t-test with Welch’s correction is used for analyses of NE and Mann-Whitney test is used for analyses of citH3 (n = 4 biological replicates). g DHL-60 cells transfected with or without pcDNA3.1-S100A12 were monitored using live cell imaging using three parameters: morphology using differential interface contrast (DIC), cell death using the cell-impermeable DNA dye SYTOX Green (green), and chromatin depolymerization using the cell-permeable DNA marker Hoechst 33342 (blue). These were performed in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were monitored every 5 min for 300 min. Important time points are shown in min. Results are representativeof at least 3 independent experiments. Scale bar, 10 μm. h, i Western blotting was conducted to detect the protein expression of NETs-related proteins and statistical results for cells transfected with pcDNA3.1-S100A12. Two-tailed unpaired t-test is used for statistical analyses of MPO, PAD4 and NE, and unpaired t-test with Welch’s correction is used for analyses of citH3 and S100A12 (n = 4 biological replicates). j Relative mRNA levels of S100A12, MPO and NE in cells transfected with pcDNA3.1-S100A12. Two-tailed unpaired t-test is used for statistical analyses of NE, and unpaired t-test with Welch’s correction is used for analyses of MPO and S100A12 (n = 4 biological replicates). k, l Representative blots of NETs-related proteins and quantification in dHL-60 cells transfected with si-S100A12. Two-tailed unpaired t-test is used for statistical analyses of MPO, PAD4, NE and citH3, and unpaired t-test with Welch’s correction is used for analyses of S100A12 (n = 4 biological replicates). m Relative mRNA levels of S100A12, MPO and NE in cells transfected with si-S100A12. Data are expressed as mean ± SEM. Statistical analyses were carried out using two-tailed unpaired t-test for (b), (d) and (m). Source data are provided as a Source Data file.

Fig. 4. Endogenous S100A12 induced neutrophil extracellular traps (NETs) formation by calcium influx overload.

a, b Intracellular calcium concentration detected by confocal Rhod2 staining, and statistical results after transfection of si-S100A12 or pcDNA3.1-S100A12 (n = 4 biological replicates). Scale bar, 10 μm. c Colorimetric detection of intracellular Ca²⁺ concentration in dHL-60 cells transfected with pcDNA3.1-S100A12 or si-S100A12 (n = 4 biological replicates). d, e Neutrophils transfected with or without si-S100A12 were preloaded with calcium probe Fluo-4 AM dye and incubated for 30 min in HBSS calcium-free media. After washing, cells were resuspended in Assay Buffer and stimulated with ionomycin (5 µM). Time-course of fluorescent signal was recorded with excitation and emission wavelengths of 488 and 526 nm, respectively. The fluorescence was normalized to the baseline fluorescence F0 (fluorescence measurement at the first time point). The Area under curve (AUC) was estimated using sums-of-squares method (n = 6 biological replicates). f–j Representative blots of peptidylarginine deiminase 4 (PAD4), p-CaMKII, myeloperoxidase (MPO), citrullination of histone 3 (citH3) and quantification in dHL-60 cells treated with pcDNA3.1-S100A12 and different concentration of calcium channel blocker (Verapamil) (n = 4 biological replicates). a: P < 0.0001 vs pcDNA3.1-NC + 0 µm Verapamil. k, l Mice were intraperitoneally injected with corn oil or Verapamil (5 mg/kg/d) every other day from Day 1 before the MI operation to post-MI Day 3. Survival curves and rates of WT and TG mice treated with Verapamil for 14 days after MI were analyzed using Log-rank (Mantel-cox) test. m, n Echocardiographic parameters were analyzed in WT and TG mice treated with verapamil at post-MI Day 28 (n = 5 biological replicates for WT MI group, n = 6 biological replicates for WT + Verapamil group, n = 3 biological replicates for TG MI group and n = 5 biological replicates for TG + Verapamil group). Data are expressed as mean ± SEM. Statistical analyses were carried out using two-tailed unpaired t-test for (m), (n), one-way ANOVA with Tukey’s multiple comparisons test for (e), (g), (h), (i), (j) and Brown-Forsythe and Welch’s ANOVA test for (b), (c). Source data are provided as a Source Data file.

Fig. 5. Annexin A5 (ANXA5) mediated endogenous S100A12-induced neutrophil extracellular traps (NETs) formation.

a Heat map of significantly differentially expressed proteins in WT and TG mice. b Relative mRNA levels of ANXA5 in BMDNs from TG and WT mice (n = 7 biological replicates). c, d Western blotting was conducted to detect the protein expression of ANXA5 for BMDNs from TG and WT mice and statistical results (n = 4 biological replicates). e Relative mRNA levels of ANXA5 in dHL-60 cells transfected with si-S100A12 or pcDNA3.1-S100A12 (n = 4 biological replicates). f, g Representative blots of ANXA5 and S100A12 and quantification in cells treated with si-S100A12, pcDNA3.1-S100A12, or rhS100A12. One-way ANOVA with Tukey’s multiple comparisons test is used for statistical analyses of ANXA5, Brown-Forsythe and Welch’s ANOVA test is used for analyses of S100A12 (n = 4 biological replicates). h, i Representative blots of myeloperoxidase (MPO), citrullination of histone 3 (citH3), ANXA5 and quantification in dHL-60 cells treated with si-ANXA5 or pcDNA3.1-ANXA5 (n = 4 biological replicates). j, k SYTOX Green staining was performed to observe the rate of NETosis in dHL-60 cells transfected with si-ANXA5 or pcDNA3.1-ANXA5 (n = 4 biological replicates). Scale bar, 10 μm. l, m Representative blots of MPO, citH3, peptidylarginine deiminase 4 (PAD4) and quantification in dHL-60 cells treated with si-100A12 and pcDNA3.1-ANXA5 (n = 4 biological replicates). One-way ANOVA with Tukey’s multiple comparisons test is used for statistical analyses of ANXA5, S100A12, MPO and citH3, Brown-Forsythe and Welch’s ANOVA test is used for analyses of PAD4. a: P < 0.01 vs si-NC + pcDNA3.1-NC, b: P < 0.01 vs si-S100A12 + pcDNA3.1-NC, c: P < 0.01 vs si-NC + pcDNA3.1-ANXA5. n, o Representative blots of MPO, citH3, PAD4 and quantification in dHL-60 cells treated with si-ANXA5 and pcDNA3.1-S100A12 with changes in exogenous S100A12 content (n = 4 biological replicates). a: P < 0.01 vs si-NC + pcDNA3.1-NC, b: P < 0.01 vs si-NC + rhS100A12, c: P < 0.01 vs si-NC + pcDNA3.1-S100A12, d: P < 0.01 vs si-ANXA5 + pcDNA3.1-NC, e: P < 0.01 vs si-ANXA5 + rhS100A12. Data are expressed as mean ± SEM. Statistical analyses were carried out using two-tailed unpaired t-test with Welch’s correction for (b), (d), one-way ANOVA with Tukey’s multiple comparisons test for (i), (k), (o) and Brown-Forsythe and Welch’s ANOVA test for (e), (c). Source data are provided as a Source Data file.

Fig. 6. Inhibition of Annexin A5 (ANXA5) expression reduced S100A12-induced neutrophil extracellular traps (NETs) formation and alleviated myocardial infarction (MI) injury.

a, b Echocardiographic parameters were analyzed in WT and TG mice pre-injected with AAV-shANXA5 or AAV-shNC at Day 3 after MI (n = 9 biological replicates for WT AAV-shNC and WT AAV-shANXA5 group, n = 7 biological replicates for TG AAV-shNC group and n = 6 biological replicates for TG AAV-shANXA5 group). c, d Echocardiographic parameters were analyzed in WT and TG mice pre-injected with AAV-shANXA5 or AAV-shNC at Day 28 after MI (n = 6 biological replicates for WT AAV-shNC group, n = 7 biological replicates for WT AAV-shANXA5 group, n = 4 biological replicates for TG AAV-shNC group and n = 3 biological replicates for TG AAV-shANXA5 group). e–g Representative blots of NETs- and apoptosis- related proteins, and quantification in WT and TG mice pre-injected with AAV-shANXA5 or AAV-shNC at Day 1 after MI (n = 4 biological replicates). h Measurements of plasma double-stranded DNA (dsDNA) concentration in WT and TG mice pre-treated with AAV-shANXA5 or AAV-shNC at Day 1 after MI (n = 4 biological replicates). i, j Immunofluorescence staining of citH3 in WT and TG mice pre-treated with AAV-shANXA5 or AAV-shNC at Day 1 after MI (n = 4 biological replicates). Scale bar, 10 μm. Data are expressed as mean ± SEM. Statistical analyses were carried out using one-way ANOVA with Tukey’s multiple comparisons test for (c), (d), (h), (j), Brown-Forsythe and Welch’s ANOVA test for (a), (g), and Kruskal-Wallis test with Dunn’s multiple-comparison test for (b). Source data are provided as a Source Data file.

Fig. 7. Correlation analysis between plasma S100A12 level and markers of neutrophil extracellular traps (NETs) in ST segment elevation myocardial infarction (STEMI) patients.

Scatter plots and regression analysis between S100A12 level and double-stranded DNA (dsDNA) concentration (a), neutrophil elastase (NE) concentration (b) and peptidylarginine deiminase 4 (PAD4) enzyme activity (c) in the plasma of STEMI patients. n = 230. Pearson’s correlation analysis was used. Source data are provided as a Source Data file.