Dapagliflozin Attenuates Post-Infarction Myocardial Fibrosis Associated With the Inhibition of NLRP3-Dependent Inflammation

Figures

• 

  Fig. 1.. Schematic overview of the in vivo experiments. (A) Flowchart of animal allocation and grouping. (B) Timeline of longitudinal in vivo imaging assessments. S, Sacrifice; positron emission tomography (PET)1: 18F-fibroblast activation protein inhibitor-74 PET; PET2: 18F-fluorodeoxyglucose PET. Abbreviations: PET, positron emission tomography.

• 

  Fig. 2.. Physiological parameters were measured at baseline and on days 7, 28, and 42 after myocardial infarction (MI). (A–C), Dapagliflozin had no significant impact on body weight (A), fasting blood glucose (B), or heart rate (C) in non-diabetic MI rats. (D,E) Dapagliflozin significantly improved both systolic blood pressure (D) and diastolic blood pressure post-MI (E). Sham and ShamD groups (n = 7); MI and MID groups (n = 9). Data are presented as means ± standard deviations (SDs). Longitudinal comparisons: mixed-effects model with Tukey’s test. Statistical significance: p < 0.05. ns: no significant difference. **** denotes statistical significance: p < 0.0001. Abbreviations: MI, myocardial infarction.

• 

  Fig. 3.. Left ventricular (LV) performance assessed by cardiac magnetic resonance on days 7 and 42 after myocardial infarction (MI). (A) Dapagliflozin improved early LV systolic function post-MI (n = 10 per group). (B) Long-term dapagliflozin treatment preserved LV systolic function and mitigated adverse cardiac remodeling following MI (Sham and ShamD groups: n = 7; MI and MID groups: n = 9). Data are presented as means ± SDs. Comparisons among three or more groups: one-way ANOVA with Tukey’s test. Statistical significance: p < 0.05. ns: no significant difference. *, **, ***, and **** denote statistical significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Abbreviations: LV, left ventricle; MI, myocardial infarction.

• 

  Fig. 4.. Evaluation of left ventricular (LV) fibrosis at Day 42 after myocardial infarction (MI). (A,B) Cardiac Masson’s trichrome staining revealed reduced LV fibrosis after dapagliflozin treatment (Sham and ShamD groups: n = 2, displayed for reference, not included in comparisons; MI and MID groups: n = 4). Scale bar = 1000 μm; original magnification: 1.5 × objective. (C,D) Real-time quantitative polymerase chain reaction (C) and western blot analysis (D) showed reduced expression of fibrosis molecular markers in the MID group rats (n = 5 per group). Data are presented as means ± SDs. Two-group comparisons: unpaired two-tailed Student’s t-test; comparisons among three or more groups: one-way ANOVA with Tukey’s test. The displayed blots were cropped for clarity and uncropped original blots are presented in Supplementary Fig. 2. Statistical significance: p < 0.05. ns: no significant difference. *, **, ***, and **** denote statistical significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Abbreviations: LV, left ventricle; MI, myocardial infarction.

• 

  Fig. 5.. Longitudinal in vivo assessment of cardiac fibroblast (CF) activation on days 6, 14, 21 and 28 after myocardial infarction (MI). (A) Representative myocardial 18F-fibroblast activation protein inhibitor-74 (18F-FAPI-74) positron emission tomography images. The red arrow indicates an 18F-fluorodeoxyglucose uptake defect in the infarcted myocardium. The white arrow indicates the 18F-FAPI-74 uptake in the infarcted myocardium and the white star indicates the tracer accumulation at the surgical incision site. (B) Dapagliflozin inhibited CF activation post-MI, as evidenced by reduced myocardial 18F-FAPI-74 uptake in the infarct region (Sham and ShamD groups: n = 7; MI and MID groups: n = 9). (C) Fibroblast activation protein expression was validated by cardiac immunohistochemistry staining (n = 5 per group). Scale bar = 100 μm; original magnification: 20 × objective. *: post-surgery. Data are presented as means ± SDs. Longitudinal comparisons: mixed-effects model with Tukey’s test; comparisons among three or more groups: one-way ANOVA with Tukey’s test. Statistical significance: p < 0.05. ns: no significant difference. *, **, ***, and **** denote statistical significance at p < 0.05,…

• 

  Fig. 6.. Effects of dapagliflozin (DAPA) on the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)-dependent signaling in infarcted cardiac tissue. (A,B) Real-time quantitative polymerase chain reaction (RT-qPCR) (A) and western blot (WB) (B) analyses demonstrated that DAPA attenuated NLRP3-dependent inflammation in infarcted cardiac tissue. (C,D) RT-qPCR (C) and WB (D) analyses demonstrated that DAPA suppressed nuclear factor kappa B pathway activation. The displayed blots were cropped for clarity and uncropped original blots are presented in Supplementary Fig. 2. n = 5 per group. Data are presented as means ± SDs. Comparisons among three or more groups: one-way ANOVA with Tukey’s test. Statistical significance: p < 0.05. ns: no significant difference. *, **, ***, and **** denote statistical significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Abbreviations: DAPA, dapagliflozin; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; RT-qPCR, real-time quantitative polymerase chain reaction; WB, western blot.

• 

  Fig. 7.. Effects of dapagliflozin (DAPA) on lipopolysaccharide (LPS)-stimulated NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation and phenotypic transition in cardiac fibroblasts (CFs) (n = 5). (A) Dose-dependent effects of LPS, DAPA, BAY 11-7082, and MCC950 on the viability of CFs. (B) DAPA reduced LPS-stimulated proliferation of CFs. (C) DAPA attenuates nuclear factor kappa B/NLRP3 pathway activation in LPS-treated CFs. (D) DAPA inhibited phenotypic transition in LPS-treated CFs. The displayed blots were cropped for clarity and uncropped original blots are presented in Supplementary Fig. 2. n = 5 per group. Comparisons among three or more groups: one-way ANOVA with Tukey’s test. Data are presented as means ± SDs. Statistical significance: p < 0.05. *, **, ***, and **** denote statistical significance vs. CON (p < 0.05, 0.01, 0.001, and 0.0001, respectively); #, ##, ###, and #### denote statistical significance vs. LPS (p < 0.05, 0.01, 0.001, and 0.0001, respectively). Abbreviations: DAPA, dapagliflozin; LPS, lipopolysaccharide; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; CFs, cardiac fibroblasts.