NKRF in Cardiac Fibroblasts Protects against Cardiac Remodeling Post‐Myocardial Infarction via Human Antigen R

Abstract Myocardial infarction (MI) remains the leading cause of death worldwide. Cardiac fibroblasts (CFs) are abundant in the heart and are responsible for cardiac repair post‐MI. NF‐κB‐repressing factor (NKRF) plays a significant role in the transcriptional inhibition of various specific genes. However, the NKRF action mechanism in CFs remains unclear in cardiac repair post‐MI. This study investigates the NKRF mechanism in cardiac remodeling and dysfunction post‐MI by establishing a CF‐specific NKRF‐knockout (NKRF‐CKO) mouse model. NKRF expression is downregulated in CFs in response to pathological cardiac remodeling in vivo and TNF‐α in vitro. NKRF‐CKO mice demonstrate worse cardiac function and survival and increased infarct size, heart weight, and MMP2 and MMP9 expression post‐MI compared with littermates. NKRF inhibits CF migration and invasion in vitro by downregulating MMP2 and MMP9 expression. Mechanistically, NKRF inhibits human antigen R (HuR) transcription by binding to the classical negative regulatory element within the HuR promoter via an NF‐κB‐dependent mechanism. This decreases HuR‐targeted M mp 2 and M mp 9 mRNA stability. This study suggests that NKRF is a therapeutic target for pathological cardiac remodeling.

Isolation of cardiac fibroblasts (CFs), cardiomyocytes (CMs), and macrophages from ischemic mouse hearts revealed a significant downregulation of NKRF expression in CFs (n = 6) in the MI group compared with that in the sham group, while no significant difference was observed in CMs (n = 6) and macrophages (n = 6).Data are the mean ± SEM.P-values were determined using unpaired two-tailed Student's ttest.NS, non-significant, *P<0.05, and **P<0.01.

Figure S2 .
Figure S2.NKRF was downregulated in CFs in a hypertensive mice model.A, Schematic diagram of the Ang II-induced hypertensive C57BL/6J mouse model.B, Picrosirius red staining shows that the ECM of the myocardium has a significant increase in collagen in hypertensive mice treated with Ang II (400 ng kg -1 min -1 ) for 4 weeks (scale bar = 50 µm).C, Immunofluorescence colocalization staining of NKRF (red) and FSP1 (green) in transverse heart cross sections obtained from C57BL/6J mice treated with Ang II (scale bar = 20 µm).ECM, extracellular matrix; CFs, cardiac fibroblasts; Ang II, angiotensin II; CTL, control; HT, hypertension; FSP1, fibroblastspecific protein 1.

Figure S4 .
Figure S4.Gene expression levels of TNF-α in the hearts of mice after myocardial infarction (MI) and sham operation.TNF-α mRNA levels were significantly elevated in the infarct border zone of the MI group (n = 5) compared to the sham group (n = 5) at 3 days post-MI.Data are presented as the mean ± SEM. ***P < 0.001 by unpaired two-tailed Student's t-test.

Figure S8 .
Figure S8.NKRF knockdown enhances TNF-α-induced expression of MMP2 and MMP9 in CFs.A and B, Verification of knockdown efficiency of three SiR-Nkrf at mRNA (A) and protein (B) levels (n = 3).C-G, Primary CFs were transfected with SiR-Nkrf or SiR-Scr for 24 h, followed by treatment with TNF-α (10 ng mL -1 ) for another 24 h.C-E, Relative expression of Nkrf (C), Mmp2 (D), and Mmp9 (E) at the mRNA level.The values were first normalized to actin and then calculated as fold changes vs. CFs transfected with SiR-Scr and treated with PBS.F, Relative expression of NKRF, MMP2, and MMP9 at the protein level.The values were first normalized to GAPDH, then calculated as fold changes vs. CFs transfected with SiR-Scr and treated with PBS.G, Representative images of gelatin zymography and quantitative analysis of MMP2 and MMP9 activities (n = 4).Samples were obtained from the supernatant of the treated CFs.Data are the mean ± SEM.P-values were determined using unpaired two-tailed Student's t-test (A and B) and two-way ANOVA with Bonferroni multiple comparisons test (C, D, E, F, and G).*P<0.05,**P<0.01,***P<0.001,and ****P<0.0001.CFs, cardiac fibroblasts; SiR-Nkrf, Nkrf siRNA (small interfering RNA); SiR-Scr, scrambled siRNA.

Figure S9 .
Figure S9.ChIP experiments using NKRF antibody enriched Mmp2 and Mmp9 gene promoter regions as templates in CFs.A, Schematic diagram of primer pairs covering the gene promoter regions of Mmp2 and Mmp9.B and C, ChIP agarose gel electrophoresis results using NKRF antibody enriched different Mmp2 (B) and Mmp9 (C) gene promoter regions as templates in CFs.CFs, cardiac fibroblasts; ChIP, chromatin immunoprecipitation.

Figure S10 .
Figure S10.Agarose gel electrophoresis results using the NRE in HuR promotor region as a template enriched by anti-NKRF, anti-p65, and anti-p50 antibody in ChIP experiments.Primary cardiac fibroblasts were pretreated with the inhibitor of the NF-κB pathway (5 M IMD 0354) for 1 h, then treated with TNF-α (10 ng mL -1 ) or PBS for another 24 h.TNF-α-induced enrichment of p65 and p50 to the NRE region within the HuR promoter was inhibited after blocking the NF-κB pathway by IMD 0354, but this did not affect the downregulation trend of NKRF enrichment in the NRE region within the HuR promoter.ChIP, chromatin immunoprecipitation; NRE, negative regulatory element.

Figure S11 .
Figure S11.HuR knockdown reverses the promoting effect of NKRF knockdown on the expression of MMP2 and MMP9 in CFs.A and B, Verification of knockdown efficiency of three SiR-HuR at mRNA (A) and protein (B) levels (n = 3).C-E, Primary CFs were transfected with SiR-Nkrf and SiR-HuR for 24 h followed by treatment with TNF-α (10 ng mL -1 ) for another 24 h.C and D, Relative expression of Nkrf, HuR, Mmp2, and Mmp9 at the mRNA (C) and protein (D) levels.The values were first normalized to actin (at the mRNA level) or GAPDH (at the protein level) and then calculated as fold changes vs. CFs transfected with SiR-Scr and treated with PBS.E, Representative images of gelatin zymography and quantitative analysis of MMP2 and MMP9 activities (n = 4).Samples were obtained from the supernatant of the treated CFs.Data are the mean ± SEM.P-values were determined using unpaired two-tailed Student's t-test (A and B) and one-way ANOVA with Bonferroni multiple comparisons test (C, D, and E).*P<0.05,**P<0.01,***P<0.001,and ****P<0.0001.CFs, cardiac fibroblasts; SiR-Nkrf, Nkrf small interfering RNA (siRNA); SiR-HuR, HuR siRNA; SiR-Scr, scrambled siRNA.

Table S1 .
Demographic and clinical information of STEMI patients and healthy donors recruited for cytokine measurement.

Table S2 .
Primer sequences used in RT-PCR.