High-Mobility Group A1 Promotes Cardiac Fibrosis by Upregulating FOXO1 in Fibroblasts

FIGURE 1. High-mobility group A1 (HMGA1) was upregulated during cardiac fibrosis. (A) The protein level of HMGA1 in mouse hearts after isoproterenol (ISO) insult (n = 6). (B) Immunohistochemical staining of HMGA1 in mouse hearts after ISO insult (n = 5). (C) The protein level of HMGA1 in fibroblasts after transforming growth factor-β1 (TGFβ1) insult (n = 6). (D) Immunofluorescence staining of fibroblasts after TGFβ1 insult (n = 5). *p < 0.05.

FIGURE 2. High-mobility group A1 (HMGA1) overexpression aggregated cardiac fibroblast activation in vitro. (A) The protein level of HMGA1 in fibroblasts transfected with ad-HMGA1 (n = 6). (B–D) Cardiac fibroblasts (CFs) were transfected with ad-HMGA1 and stimulated with TGFβ1 for 24 h. (B) Cell proliferation detected by cell counting kit 8 (CCK8) assay (n = 6). (C) Immunofluorescence staining of PCNA (n = 5). (D) Immunofluorescence staining of α-SMA (n = 5). (E) Transcription levels of collagen I, collagen III, and connective transforming tissue factor (CTGF) (n = 6). *p < 0.05.

FIGURE 3. High-mobility group A1 (HMGA1) overexpression contributed to ISO-induced cardiac fibrosis in vivo. (A) The protein level of HMGA1 in mouse hearts 3 weeks after AAV9-HMGA1 injection (n = 6). (B) Immunofluorescence staining of HMGA1 and α-SMA in heart tissue post-AAV9-HMGA1 injection and ISO injection (n = 5). (C) Picrosirius red (PSR) staining image and quantitative result of left ventricular collagen volume in mouse hearts after ISO treatment (n = 5). (D) Transcription levels of collagen I, collagen III, and TGFβ1 (n = 6). (E) Protein levels of MMP9, collagen III, and α-SMA in heart tissue (n = 6). (F) Echocardiographic results in each group (n = 10). *p < 0.05.

FIGURE 4. High-mobility group A1 (HMGA1) deficiency reduced cardiac fibroblast proliferation and activation in vitro. (A) The protein level of HMGA1 in fibroblasts transfected with HMGA1 siRNA (n = 6). (B–D) CFs were transfected with HMGA1 siRNA and stimulated with TGFβ1 for 24 h. (B) Cell proliferation detected by CCK8 assay (n = 6). (C) Immunofluorescence staining of PCNA (n = 5). (D) Immunofluorescence staining of α-SMA (n = 5). (E) Transcription levels of collagen I, collagen III, and CTGF (n = 6). *p < 0.05.

FIGURE 5. High-mobility group A1 (HMGA1) modulated the expression and phosphorylation of FOXO1. (A) The protein level of FOXO1 in heart tissue after AAV9-HMGA1 and ISO injection (n = 6). (B) The protein level of FOXO1 in CFs after HMGA1 siRNA and TGFβ1 treatment (n = 6). (C) The mRNA level of FOXO1 in heart tissue (left) and CFs (right) in each group (n = 6). (D) Luciferase reporter results in CFs with HMGA1 overexpression or HMGA1 silencing. *p < 0.05.

FIGURE 6. FOXO1 inhibition abolished the effect of HMGA1 overexpression in vitro. CFs were treated with AS1842856 (10 μM) and then treated with TGFβ1 for 24 h. (A) Cell proliferation detected by CCK8 assay (n = 6). (B) Immunofluorescence staining of α-SMA (n = 5). (C) Immunofluorescence staining of PCNA (n = 5). (D) Transcription levels of collagen I, collagen III, and CTGF (n = 6). (E) Protein levels of collagen III and α-SMA (n = 6). *p < 0.05.

FIGURE 7. High-mobility group A1 (HMGA1) knockdown alleviated cardiac fibrosis and dysfunction induced by angiotensin II (Ang II) in mice. (A) The protein level of HMGA1 in mouse hearts 3 weeks after AAV9-shHMGA1 injection (n = 6). (B) Immunofluorescence staining of HMGA1 and α-SMA in heart tissue post-AAV9-shHMGA1 injection and Ang II infusion (n = 5). (C) PSR staining image and quantitative result of left ventricular collagen volume in mouse hearts 28 days after Ang II infusion (n = 5). (D) Transcription levels of collagen I, collagen III, and TGFβ1 (n = 6). (E) Protein levels of collagen III and α-SMA in heart tissue (n = 6). (F) Echocardiographic results in each group (n = 10). *p < 0.05.

FIGURE 8. FOXO1 inhibition attenuated HMGA1-induced cardiac dysfunction. (A) The protein level of FOXO1 in mouse hearts 3 weeks after AAV9-shFOXO1 injection (n = 6). (B) PSR staining image and quantitative result of left ventricular collagen volume in mouse hearts 28 days after Ang II infusion (n = 5). (C) Transcription levels of collagen I, collagen III, and TGFβ1 (n = 6). (D) Protein levels of collagen III and α-SMA in heart tissue (n = 6). (E) Echocardiographic results in each group (n = 10). *p < 0.05.