The effect of HMGA1 in LPS-induced Myocardial Inflammation

Abstract

Aims: The High Mobility Group A1 (HMGA1) proteins, serving as a dynamic regulator of gene transcription and chromatin remodeling, play an influential part in the pathological process of a large number of cardiovascular diseases. However, the precise role of HMGA1 in sepsis induced cardiomyopathy (SIC) remains unintelligible. This research was designed to illustrate the effect of HMGA1 involved in SIC. Methods and Results: Cardiomyocyte-specific HMGA1 overexpression was obtained using an adeno-associated virus system with intramyocardial injection in mice heart. The model of SIC in mice was constructed via intraperitoneal injection of lipopolysaccharide (LPS) for 6h. H9c2 rat cardiomyocytes was stimulated with LPS for 12h. HMGA1 expression was upregulated in murine inflammatory hearts as well as LPS stimulated H9c2 cardiomyocytes. HMGA1-overexpressing exhibited aggravated cardiac dysfunction, cardiac inflammation as well as cells apoptosis following LPS treatment both in vivo and in vitro experiment. Interestingly, HMGA1 knockdown in H9c2 cardiomyocytes attenuated LPS-induced cardiomyocyte inflammation, but aggravated cell apoptosis. Mechanistically, we found that overexpression of HMGA1 induced increased expression of cyclooxygenase-2 (COX-2). COX-2 inhibitor alleviated the aggravation of inflammation and apoptosis in HMGA1 overexpressed H9c2 cardiomyocytes whereas HMGA1 knockdown induced a reduction in signal transducer and activators of transcription 3 (STAT3) expression. STAT3 agonist reversed HMGA1 silence induced anti-inflammatory effects, while ameliorated cell apoptosis induced by LPS. Conclusion: In conclusion, our results suggest that overexpression of HMGA1 aggravated cardiomyocytes inflammation and apoptosis by up-regulating COX-2 expression, while silence of HMGA1 expression attenuated inflammation but aggregated cell apoptosis via down-regulation of STAT3.

Keywords: HMGA1; Myocardial inflammation; STAT3; cyclooxygenase-2; lipopolysaccharide.

Figure 1

HMGA1 expression of heart tissue or cardiomyocytes up-regulated after LPS stimuli. (A) Protein expression level of HMGA1 in mice heart with or without LPS stimuli was shown in western blotting and was normalized to GAPDH (n=6). (B) Representative images of western blotting of HMGA1 expression in H9c2 cardiomyocytes and the protein expression level was normalized to GAPDH (n=6). (C) Cardiac HMGA1 mRNA expression was detected by PCR (n=6). (D) HMGA1 mRNA expression of H9c2 cardiomyocytes was examined by PCR (n=6). (E) HMGA1 expression in heart tissue was detected by immunohistochemistry staining (n=6). (F) HMGA1 expression in H9c2 cardiomyocytes was revealed by immunofluorescence staining (n=6). *P<0.05, vs. control group. Data were analyzed using Student's t-test.

Figure 2

HMGA1 promoted cardiomyocytes inflammation and apoptosis induced by LPS. (A) The protein expression level of HMGA1 in the H9c2 cardiomyocytes following adeno-associated virus infection was normalized to GAPDH (n=6). (B) The viability of each group was detected by CCK8 (n=6). (C) RT-PCR for mRNA expression of inflammatory cytokines in H9c2 cardiomyocytes, such as TNF-α, IL-1, IL-6 (n=6). (D) TUNEL staining exhibited the cellular apoptosis in H9c2 cardiomyocytes after infection of AAV9-GFP or AAV9-HMGA1 vector with or without LPS stimuli (n=6). (E) Western blotting analyses of the apoptosis-related proteins, including Bax, Bcl-2 and Cytochrome C. and the protein expression level was normalized to GAPDH (n=6). *P<0.05, vs. AAV9-GFP+PBS group. ^#P<0.05, vs. AAV9-GFP+ LPS group. The data are expressed as the mean±standard deviation (SD) and were compared by one-way ANOVA with Tukey post hoc analysis.

Figure 3

Cardiac HMGA1-overexpression mice subjected to the treatment of LPS exhibited cardiac dysfunction. (A) Immunoblots for HMGA1 and GAPDH following intramyocardial injection of AAV9-GFP or AAV9-HMGA1 vector(n=6) and HMGA1 expression was normalized to GAPDH.(B) Echocardiographic analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and heart rate(HR) 6h after injection of saline or LPS(n=10). (C) RT-PCR for mRNA level of inflammatory related genes in heart tissue, such as TNF-α, IL-1, IL-6 (n=6). (D) TUNEL staining showed the level of cell apoptosis in mouse heart tissue. (E) Representative western blotting images of the apoptosis-related proteins, including BAX, Bcl-2 and Cytochrome C. and the protein expression levels were normalized to GAPDH (n=6). *P<0.05, vs. CON group. ^#P<0.05, vs. LPS group. The data are expressed as the mean±SD and were compared by one-way ANOVA with Tukey post hoc analysis.

Figure 4

HMGA1 deficiency reduced cardiomyocyte inflammation, but aggravated apoptosis. (A) HMGA1 expression in the H9c2 cardiomyocytes after transfection with si-HMGA1 to knock down HMGA1 and protein expression level was normalized to GAPDH (n=6). (B) The viability of each group was detected by CCK8 (repeat 6 times). (C) RT-PCR for mRNA level of inflammatory cytokines in H9c2 cardiomyocytes, such as TNF-α, IL-1, IL-6 (n=6). (D) TUNEL staining exhibited the cellular apoptosis in H9c2 cardiomyocytes after transfection with siRNA or siHMGA1 with or without stimulation of LPS (n=6). (E) Western blotting analyses of the apoptosis-related proteins in H9c2 cardiomyocytes, such as Bax, Bcl-2 and Cytochrome C. and the protein expression level were normalized to GAPDH(n=6). *P<0.05, vs. si-NC+PBS group. ^#P<0.05, vs. si-NC+LPS group. The data are expressed as the mean±SD and were compared by one-way ANOVA with Tukey post hoc analysis.

Figure 5

HMGA1 altered the expression of COX-2 and STAT3. (A) Representative western blotting images of P-STAT, STAT3 and COX-2 expression of H9c2 cardiomyocytes after infection of AAV9-GFP or AAV9-HMGA1 vector with or without LPS stimulation and the protein expression level was normalized to GAPDH(n=6). (B) RT-PCR for mRNA level of STAT3 and COX-2 after infection of AAV9-GFP or AAV9-HMGA1 vector with or without LPS stimuli and the expression level was normalized to GAPDH(n=6). (C) Representative western blotting images of P-STAT3, STAT3 and COX-2 expression of H9c2 cardiomyocytes after transfection of si-NC or si-HMGA1 with or without LPS treatment and all of the proteins were normalized to GAPDH(n=6). (D) RT-PCR for mRNA level of STAT3 and COX-2 after infection of si-HMGA1 or si-NC with or without LPS stimuli (n=6). *P<0.05, vs. AAV9-GFP/si-NC+PBS group. ^#P<0.05, vs. AAV9-GFP/si-NC+LPS group. The data are represented as the mean±SD and compared by one-way ANOVA with Tukey post hoc analysis.

Figure 6

COX-2 deletion abolished the effect of HMGA1 overexpression and STAT3 agonist reversed the role of HMGA1 silence. (A-C): H9c2 cardiomyocytes were transfected with AAV9-HMGA1 then stimulated with LPS and treated with celecoxib (COX-2 inhibitor). (A)RT-PCR analyses for transcriptional level of inflammatory cytokines (TNF-α, IL-1, IL-6) in each group (n = 6). (B)TUNEL staining results for the detection of the apoptosis-positive cells in each group (n =6). (C) The protein expression level of pro-apoptotic cytochrome C was examined by western blotting and was normalized to GAPDH (n=6). *P<0.05 vs. the LPS group; ^#P<0.05 vs. the AAV9-HMGA1+LPS group. The data were shown as the mean±SD and were compared by one-way ANOVA with Tukey post hoc analysis. (D-F): H9c2 cardiomyocytes were processed with siHMGA1 then stimulated with LPS and treated with colivelin (a STAT3 agonist, 1uM). (D) RT-PCR analyses for transcriptional level of inflammatory cytokines (TNF-α, IL-1, IL-6) in each group (n = 6). (E)The apoptotic cells in each group were detected by TUNEL staining (n =6). (F) Western blotting result for the detection of cytochrome C and the expression level was normalized to GAPDH in each group (n=6). *P<0.05 vs. the LPS group; #P<0.05 vs. the si-HMGA1+LPS group. The data were shown as the mean±SD and were compared by one-way ANOVA with Tukey post hoc analysis.