Empagliflozin Limits Myocardial Infarction in Vivo and Cell Death in Vitro: Role of STAT3, Mitochondria, and Redox Aspects

Abstract

Empagliflozin (EMPA), a drug approved for type 2 diabetes management, reduced cardiovascular death but is unknown if it reduces myocardial infarction. We sought to investigate: (i) the effect of EMPA on myocardial function and infarct size after ischemia/reperfusion in mice fed with western diet (WD), (ii) the underlying signaling pathways, (iii) its effects on cell survival in rat embryonic-heart-derived cardiomyoblasts (H9C2) and endothelial cells (ECs). To facilitate the aforementioned aims, mice were initially randomized in Control and EMPA groups and were subjected to 30 min ischemia and 2 h reperfusion. EMPA reduced body weight, blood glucose levels, and mean arterial pressure. Cholesterol, triglyceride, and AGEs remained unchanged. Left ventricular fractional shortening was improved (43.97 ± 0.92 vs. 40.75 ± 0.61%) and infarct size reduced (33.2 ± 0.01 vs. 17.6 ± 0.02%). In a second series of experiments, mice were subjected to the above interventions up to the 10th min of reperfusion and myocardial biopsies were obtained for assessment of the signaling cascade. STAT3 was increased in parallel with reduced levels of malondialdehyde (MDA) and reduced expression of myocardial iNOS and interleukin-6. Cell viability and ATP content were increased in H9C2 and in ECs. While, STAT3 phosphorylation is known to bestow infarct sparing properties through interaction with mitochondria, we observed that EMPA did not directly alter the mitochondrial calcium retention capacity (CRC); therefore, its effect in reducing myocardial infarction is STAT3 dependent. In conclusion, EMPA improves myocardial function and reduces infarct size as well as improves redox regulation by decreasing iNOS expression and subsequently lipid peroxidation as shown by its surrogate marker MDA. The mechanisms of action implicate the activation of STAT3 anti-oxidant and anti-inflammatory properties.

Keywords: STAT3 pathway; cardiac function; cardioprotection; empagliflozin; infarct size; molecular signaling.

Figure 1

Empagliflozin reduces body weight, glucose levels, and lipid peroxidation levels without altering AGE products. (A) Experimental work flow. (B) Effects of diet manipulation and empagliflozin treatment on mice BW (^*p < 0.05 vs. Baseline, ^#p < 0.05 vs. Control). (C) Effects of diet manipulation and empagliflozin treatment on fasting glucose levels (^*p < 0.05 vs. Baseline, ^#p < 0.05 vs. Control). (D) AGE (AU) measured as Intergrated Fluorescence at emission 370 nm and (E) effects of diet manipulation and empagliflozin treatment on circulating MDA (uM) levels (^*p < 0.05 vs. Control).

Figure 2

Empagliflozin pretreatment reduces myocardial infarct size in mice treated with WD. (A) Representative Graphs of Infarct/Risk Area (I/R) %. Individual animals represented as a scatter plot with dots in the Control group and squares in the EMPA group. Results plotted as Mean ± SEM (^*p < 0.05 vs. Control). (B) Risk/All Area (R/A) %.

Figure 3

Empagliflozin induces cardioprotection through activation of STAT-3 and independently of RISK pathway and of AMPK activation. Representative western blots and relative densitometry graphs of (A) p-Akt (S473)/t-Akt and t-Akt/GAPDH (B) p-eNOS(S1177)/t-eNOS and t-eNOS/GAPDH (C) p-GSK-3β(S9)/t-GSK-3β and t-GSK-3β/GAPDH (D) p-ERK1/2 (Thr202/Tyr204)/t-ERK1/2 and t-ERK1/2/GAPDH (E) p-AMPKα(S172)/t-AMPKα and t-AMPKα/GAPDH (F) p-STAT3(Tyr705)/t-STAT3 and t-STAT3/β-actin (^*p < 0.05 vs. Control). p-Akt, t-Akt (A) and p-ERK1/2, t-ERK1/2 (D) proteins were detected on the same gel and share the same image of GAPDH, serving as loading Control. This was also the case for p-eNOS, t-eNOS (B), p-AMPKα, and t-AMPKα (E).

Figure 4

Empagliflozin reduces myocardial iNOS and IL-6 expression. Representative western blots and relative densitometry graphs of (A) p-NF-κB (p65) (S536)/ t-NF-κB (p65) and t-NF-κB (p65)/β-tubulin (B) IL-6/ β-tubulin (^*p < 0.05 vs. Control) and (C) iNOS/ β-tubulin (^*p < 0.05 vs. Control). p-NFκB(p65), t-NFkB(p65) (A) and IL-6 (B) were detected on the same gel and share the same image of β tubulin, serving as loading Control.

Figure 5

Evaluation of the effects of EMPA on isolated heart mitochondria. Direct effects of empagliflozin on mitochondria. (A) Calcium retention capacity of mouse heart mitochondria (^***p < 0.001 vs. all other study groups) and (B) representative Calcium tracing (n = 5).

Figure 6

Empagliflozin rescues ECs and H9C2 cells from hypoxia/reoxygenation injury. (A,B) MTT assay was used to assess the effect of EMPA in hypoxia/reoxygenation setting. The indicated concentrations were used to treat H9C2 cells (A) and ECs (B). Both H9C2 and ECs, either untreated or treated with AGE (1 mg/mL), were subjected to hypoxia/reoxygenation. Data normalized to control are reported as mean ± SD and representative of four different experiments performed in triplicate (n = 12) (For H9C2, ^***p < 0.001 EMPA 500 nM vs. control and EMPA 100 nM; ^**p < 0.01 EMPA 500 nM + AGE vs. control + AGE and EMPA 100 nM + AGE; for ECs, ^**p < 0.01 EMPA 500 nM vs. control and EMPA 100 nM; ^*p < 0.05 EMPA 500 nM + AGE vs. control + AGE. (C,D) Histogram representation of the relative cellular ATP content. Data are obtained from H9C2 cells and ECs treated as indicated (for H9C2, ^*p < 0.05 EMPA 500 nM vs. control; ^***p < 0.001 EMPA 500 nM + AGE vs. control + AGE and EMPA 100 nM + AGE; for ECs, ^***p < 0.001 EMPA 500 nM vs. control and EMPA 100 nM; ^**p < 0.01 control + AGE vs. EMPA 100 nM + AGE, EMPA 500 nM + AGE vs. control + AGE, ^***p < 0.001 EMPA 500 nM + AGE vs. EMPA 100 nM + AGE). Data are reported as mean ± SD and representative of four different experiments performed in triplicate (n = 12). (E) Cell extracts from H9C2 cells treated, with or without AGE (1 mg/mL), and with Empagliflozin at the indicated concentrations, were subjected to hypoxia/reoxygenation and analyzed for RAGE content, and normalized to β-actin Data are representative of four different experiments performed in triplicate (n = 12).