Transglutaminase 2 inhibition ameliorates cardiac fibrosis in myocardial infarction by inducing M2 macrophage polarization in vitro and in vivo

Abstract

Objective: Macrophages perform vital functions in cardiac remodeling after myocardial infarction (MI). Transglutaminase 2 (TG2) participates in fibrosis. Nevertheless, the role of TG2 in MI and mechanisms underlying macrophage polarization are unclear. This study aimed to discover the functions and possible mechanisms of TG2 in MI.

Material and methods: C57BL/6 mice were classified into three groups (six mice per group): Sham, MI, and MI+GK921 groups. GK921 acts as a TG2 inhibitor. Cardiac function, myocardial cell apoptosis, fibrosis, and macrophage phenotype in mouse experiments were detected through echocardiography, terminal deoxynucleotidyl transferase dUTP nick end labeling, Masson staining, immunofluorescence, and flow cytometry, respectively. The in vitro study involved the treatment of mouse cardiac fibroblasts isolated from mice with transforming growth factor β1 (TGF-β1) and evaluation of fibrosis through the detection of the expressions of fibrosis-associated proteins using Western blot. Bone marrow-derived macrophages (BMDMs) obtained from mice were triggered by interleukin (IL)-4, and the type of macrophages was determined through flow cytometry.

Results: In in vivo experiments, GK921 substantially improved cardiac injury and fibrosis, induced M2 macrophage polarization, and suppressed the TGF-β1/small mother against decapentaplegic 3 (Smad3) pathway in MI mice. Moreover, TG2 knockdown considerably decreased the expressions of fibrosis-associated proteins in TGF-β1-triggered mouse cardiac fibroblasts, which indicates the repressive effect of TG2 knockdown on fibrosis. In addition, the inhibition effect of TG2 downregulation on the TGF-β1/Smad3 pathway was proven in TGF-β1-treated mouse cardiac fibroblasts in vitro. Moreover, TG2 inhibition remarkably increased M2 macrophage polarization in IL-4-induced BMDMs.

Conclusion: TG2 inhibition facilitated M2 macrophage polarization to provide protection against MI-caused cardiac fibrosis in mice, and these effects may be attained through modulation of the TGF-β1/Smad3 pathway.

Keywords: Macrophage polarization; Myocardial infarction; Transforming growth factor β1/small mother against decapentaplegic 3 pathway; Transglutaminase 2.

Figure 1:

Transglutaminase 2 inhibition improved myocardial infarction (MI)-induced cardiac dysfunction and myocardial damage. Myocardial infarction model was formed using C57BL/6 mice, and GK921 (a transglutaminase 2 inhibitor) was applied in mouse pre-treatment before myocardial infarction. (a-e) Echocardiography was performed, and cardiac function parameters, including left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular volume at end diastole (LV vol: s), and Left ventricular internal dimension in systole (LVIDs), were detected (n = 6). (f-g) Cardiomyocyte apoptosis was determined through Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (n = 3). Scale bar, 100 µm. TUNEL staining shows green fluorescence, the cell nucleus are stained with 4’,6-diamidino-2-phenylindole (DAPI) and show blue fluorescence. (h-i) Masson’s trichrome staining was used to evaluate cardiac fibrosis in mice (n = 3). (Scale bar, 100 µm. *P < 0.05, and ***P < 0.001. DAPI: 4’,6-diamidino-2-phenylindole.)

Figure 2:

Transglutaminase 2 inhibition promoted M2 macrophage polarization in mice after myocardial infarction (MI). (a and b) Flow cytometry was performed to assess myocardial epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80)+ macrophage mannose receptor (CD206)+ macrophages (n = 3). (c-e) Immunofluorescence staining of M2 macrophage polarization markers in mouse heart tissues (n = 3). Scale bar, 100 µm. The CD206 and arginase-1 (Arg-1) staining exhibits red fluorescence, and 4’,6-diamidino-2-phenylindole (DAPI) staining for labeling cell nuclei shows blue fluorescence. (f and g) Protein expressions of CD206 and Arg-1 in mouse heart tissues were detected using Western blot (n = 3). (*P < 0.05, **P < 0.01, and ***P < 0.001. DAPI: 4’,6-diamidino-2-phenylindole, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.)

Figure 3:

Transglutaminase 2 inhibition suppressed the transforming growth factor (TGF)-β1/Small mother against decapentaplegic 3 (Smad3) pathway in myocardial infarction (MI) mice. (a) Transglutaminase 2 (TG2), TGF-β1, phosphoSmad3 (p-Smad3), and Smad3 protein expression measured in mouse heart tissues via Western blot (n = 3). (b - d) Quantitative analysis of TG2, TGF-β1, p-Smad3/Smad3 (n = 3). (*P < 0.05. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.)

Figure 4:

Transglutaminase 2 downregulation transforming growth factor (TGF)-β1-induced myocardial fibrosis and repressed TGF-β1/small mother against decapentaplegic 3 (Smad3) pathway in mouse cardiac fibroblasts. (a and b) Small interfering negative control (si-NC) and siTransglutaminase 2 (si-TG2) were transfected to mouse cardiac fibroblasts isolated from C57BL/6 mice, and the protein expression of TG2 was detected through Western blot. (c-f) Mouse cardiac fibroblasts were treated with 100 µg/mL TGF-β1 to induce fibrosis, followed by transfection with siNC and si-TG2 for 48 h. Western blot was conducted to determine the levels of fibrosis-associated proteins, including collagen I, collagen III, and α-smooth muscle actin (α-SMA), in mouse cardiac fibroblasts after TGF-β1 treatment and transfection (n = 3). (g-i) Western blot was also used in the assessment of the levels of TGF-β1, phospho-Smad3 (p-Smad3), and Smad3 in mouse cardiac fibroblasts after TGF-β1 treatment and transfection (n = 3). (*P < 0.05. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.)

Figure 5:

Transglutaminase 2 inhibition increased M2 macrophage polarization in interleukin (IL)-4-induced bone marrow derived macrophages. (a-d) Western blot was utilized to assess the levels of macrophage mannose receptor (CD206), arginase-1 (Arg-1) and interleukin-10 (IL-10) in interleukin (IL)-4-induced bone marrow derived macrophages after transfection of small interfering negative control (si-NC) and si-Transglutaminase 2 (si-TG2) (n = 3). (*P < 0.05. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.)