Signal Transducer and Activator of Transcription-3 Modulation of Cardiac Pathology in Chronic Chagasic Cardiomyopathy

Abstract

Chronic Chagasic cardiomyopathy (CCC) is a severe clinical manifestation that develops in 30%-40% of individuals chronically infected with the protozoal parasite Trypanosoma cruzi and is thus an important public health problem. Parasite persistence during chronic infection drives pathologic changes in the heart, including myocardial inflammation and progressive fibrosis, that contribute to clinical disease. Clinical manifestations of CCC span a range of symptoms, including cardiac arrhythmias, thromboembolic disease, dilated cardiomyopathy, and heart failure. This study aimed to investigate the role of signal transducer and activator of transcription-3 (STAT3) in cardiac pathology in a mouse model of CCC. STAT3 is a known cellular mediator of collagen deposition and fibrosis. Mice were infected with T. cruzi and then treated daily from 70 to 91 days post infection (DPI) with TTI-101, a small molecule inhibitor of STAT3; benznidazole; a combination of benznidazole and TTI-101; or vehicle alone. Cardiac function was evaluated at the beginning and end of treatment by echocardiography. By the end of treatment, STAT3 inhibition with TTI-101 eliminated cardiac fibrosis and fibrosis biomarkers but increased cardiac inflammation; serum levels of interleukin-6 (IL-6), and IFN-γ; cardiac gene expression of STAT1 and nuclear factor-κB (NF-κB); and upregulation of IL-6 and Type I and Type II IFN responses. Concurrently, decreased heart function was measured by echocardiography and myocardial strain. These results indicate that STAT3 plays a critical role in the cardiac inflammatory-fibrotic axis during CCC.

Keywords: STAT3; Trypanosoma cruzi; chronic Chagasic cardiomyopathy; fibrosis; inflammation.

Figure 1

Experimental design. Shown is the timeline for experimental infection with Trypanosoma cruzi, TTI-101 and/or benznidazole treatment, and echocardiography imaging in a mouse model of chronic Chagasic cardiomyopathy (A). Six treatment and control groups are listed, with untreated animals receiving dimethyl sulfoxide (DMSO) treatment vehicle alone (B).

Figure 2

Active signal transducer and activator of transcription-3 (STAT3) concentration in cardiac fibroblast cell culture lysate. Shown are phosphorylated STAT3 (pY-STAT3) concentrations (units/µl, each µl contained 100 µg protein) in cardiac cell culture lysate. Separate cultures of cardiac fibroblasts were infected with one trypomastigote per cell of H1 TCI Trypanosoma cruzi strain or left naive. One each from the infected and naive groups was treated with TTI-101 (20 µM) or media alone. Cells were lysed at 12, 24, 48, and 72 h post infection. Cell lysates were analyzed with ELISA for pY-STAT3 concentration. Depicted are the summary data from two biological and two technical replicates. Error bars represent SD. Data were analyzed with two-way ANOVA; ****p < 0.0001 when comparing TTI-101-treated cells (purple symbols) to infected untreated cells (green symbols), ^$$$$p < 0.0001 when comparing TTI-101-treated cells (purple symbols) to naive untreated cells (blue symbols).

Figure 3

Cardiac parasite burden and signal transducer and activator of transcription-3 (STAT3) activity. Shown are the mean ± SD concentrations of pY-STAT3 measured in cardiac tissue (A) and the relative parasite burden per 10 mg cardiac tissue measured with qPCR at 91 days post infection (B). Data were analyzed with Student’s t-test; *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

Cardiac fibrosis and fibrosis biomarkers in experimental animals. Relative fibrosis % of total cardiac tissue imaged is illustrated for 91 days post infection (DPI) at conclusion of treatment (A). Terminal serum from all animals was collected at the conclusion of treatment at 91 DPI. Biomarkers of cardiac fibrosis were measured by ELISA. Serum concentrations of transforming growth factor beta (TGF-β) (B) and platelet-derived growth factor (PDGF)-D (C) at 91 DPI are shown. Representative images from one of two replicate experiments are shown. Error bars represent SD. Data were analyzed with Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Cardiac inflammation quantified on histopathological analysis in experimental animals. Representative images of hematoxylin and eosin staining of infiltrating inflammatory cells in naive untreated (A), naive TTI-101 treated (B), infected untreated (C), infected benznidazole treated (D), infected TTI-101 treated (E), and infected TTI-101 with benznidazole treated (F) are shown. Relative inflammation % of total cardiac tissue imaged is illustrated for 91 days post infection, at the conclusion of treatment (G). Representative images from one of two replicate experiments are shown. Error bars represent SD. Data were analyzed with Student’s t-test; *p < 0.05, ***p < 0.001, **** p < 0.0001.

Figure 6

CD4 and CD8 staining of inflammatory infiltrate in cardiac tissue. Immunohistochemistry was performed on cardiac tissues from groups exhibiting myocarditis, and images were processed for the measurement of relative CD4+ and CD8+ populations. The relative CD4+ (A) and CD8+ (B) stained cells are depicted as a percent of total tissue, measured from heart samples from all treatment groups. Comparison of CD4+ and CD8+ percentages of total cellular infiltrate (C) is shown. Error bars represent SD. Data were analyzed with Student’s t-test; ***p < 0.001, ****p < 0.0001.

Figure 7

Inflammatory signaling induced by signal transducer and activator of transcription-3 (STAT3) inhibition. Pro-inflammatory signaling mechanisms influenced by STAT3 activity were measured in the serum and cardiac tissues of all animals after treatment with TTI-101. Shown are individual levels of serum interleukin-6 (IL-6) (A), cardiac IL-6 (B), and serum interferon-γ (IFN-γ) (C). Downstream effectors of IL-6 and IFN-γ were measured in cardiac tissue. Cardiac gene expression levels of STAT1 (D) and NF-κB (E) are shown as fold increase above naive controls. Representative images from one of two replicate experiments are shown. Error bars represent SD. Data were analyzed with Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 8

Cardiac function measured on echocardiography. Echocardiography was performed pre- and post-treatment to evaluate changes to cardiac function. All infected groups are compared to the naive untreated and naive TTI-101-treated groups (no significant differences were seen between the naive untreated and naive TTI-101-treated groups). Summary data measurements of ejection fraction (A) and cardiac output (B) measured by M mode echocardiography before and after treatment compared to naive control averages (dotted line). Summary data levels of myocardial strain of the left ventricle measured by B mode echocardiography before and after treatment compared to naive control average (dotted line; (C). Short-axis images were used to determine myocardial circumferential strain (C). Representative images from one of two replicate experiments are shown. Error bars represent SD. Data were analyzed with Student’s t-test. When comparing groups to naive control, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For each group when comparing pre-treatment to post-treatment values ^±p < 0.05, ^±±p < 0.01, ^±±±±p < 0.0001.

Figure 9

Signal transducer and activator of transcription-3 (STAT3) activity in fibrosis and inflammatory signaling. Depicted are the pro-inflammatory and pro-fibrotic pathways in which STAT3 plays a role. Arrows represent upregulation of a pathway, while blunt lines represent downregulation of a pathway. Cardiac inflammation is modulated by STAT3 via suppression of both nuclear factor-κB (NF-κB) and STAT1 pro-inflammatory signaling. Cardiac fibrosis signaling is promoted by STAT3 through transforming growth factor beta (TGF-β)/SMAD signaling. The image was created with the assistance of BioRender.

Figure 10

Signal transducer and activator of transcription-3 (STAT3) modulation of the cardiac inflammatory–fibrotic axis in chronic Chagasic cardiomyopathy (CCC). Depicted is the cardiac pathology continuum that starts in the acute phase with a predominantly inflammatory response in the heart, which develops into a predominantly fibrotic response in the heart with progression into chronic disease. The role of STAT3 and its upstream and downstream signaling mediators is also depicted. In our model of CCC, STAT3 was found to promote fibrosis via transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF) signaling while suppressing inflammatory signaling that involved interferon-γ (IFN-γ) and interleukin-6 (IL-6). Upon inhibition of STAT3, inflammation increased and fibrosis decreased in the heart. The image was created with the assistance of BioRender.