Empagliflozin attenuates cardiac microvascular ischemia/reperfusion through activating the AMPKα1/ULK1/FUNDC1/mitophagy pathway

Abstract

Mitophagy preserves microvascular structure and function during myocardial ischemia/reperfusion (I/R) injury. Empagliflozin, an anti-diabetes drug, may also protect mitochondria. We explored whether empagliflozin could reduce cardiac microvascular I/R injury by enhancing mitophagy. In mice, I/R injury induced luminal stenosis, microvessel wall damage, erythrocyte accumulation and perfusion defects in the myocardial microcirculation. Additionally, I/R triggered endothelial hyperpermeability and myocardial neutrophil infiltration, which upregulated adhesive factors and endothelin-1 but downregulated vascular endothelial cadherin and endothelial nitric oxide synthase in heart tissue. In vitro, I/R impaired the endothelial barrier function and integrity of cardiac microvascular endothelial cells (CMECs), while empagliflozin preserved CMEC homeostasis and thus maintained cardiac microvascular structure and function. I/R activated mitochondrial fission, oxidative stress and apoptotic signaling in CMECs, whereas empagliflozin normalized mitochondrial fission and fusion, neutralized supraphysiologic reactive oxygen species concentrations and suppressed mitochondrial apoptosis. Empagliflozin exerted these protective effects by activating FUNDC1-dependent mitophagy through the AMPKα1/ULK1 pathway. Both in vitro and in vivo, genetic ablation of AMPKα1 or FUNDC1 abolished the beneficial effects of empagliflozin on the myocardial microvasculature and CMECs. Taken together, the preservation of mitochondrial function through an activation of the AMPKα1/ULK1/FUNDC1/mitophagy pathway is the working mechanism of empagliflozin in attenuating cardiac microvascular I/R injury.

Keywords: AMPKα1/ULK1 pathway; Cardiac microvascular I/R injury; Empagliflozin; FUNDC1-Dependent mitophagy.

Graphical abstract

Fig. 1

Empagliflozin attenuates I/R-induced cardiac microvascular damage. Mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. (A) An electron microscope was used to detect changes in the microvascular structure of the reperfused heart. Yellow arrows indicate microvascular endothelial cell inflation and luminal stenosis. (B) Heart tissue was stained using H&E to visualize the morphology of erythrocytes. Black arrows indicate erythrocyte accumulation in microvessels. (C–D) Double immunofluorescence staining of Gr1⁺ neutrophils and troponin T (TnT)⁺ cardiomyocytes. DAPI was used to visualize the nucleus in the myocardium. (E–G) RNA was isolated from heart tissues, and qRT-PCR was used to determine the expression of inflammatory cytokines (TNFα, MCP1 and IL-6). Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Empagliflozin alleviates I/R-induced CMEC damage.In vivo, mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts. The cells were cultured for 24 h and then used for functional analyses. (A–C)In vivo, proteins were isolated from reperfused heart tissues, and Western blots were used to analyze eNOS and ET-1 levels. (D, E)In vivo, immunofluorescence staining of VE-cadherin was performed to observe the changes in endothelial integrity and barrier function. (F, G) Immunohistochemistry was used to assess the expression of ICAM1, an endothelium-specific adhesive factor. (H–J) Proteins were isolated from CMECs, and the levels of Src and Fak were analyzed through Western blots. (K, L)In vitro, FITC-dextran clearance and TER assays were used to analyze the endothelial barrier function and integrity of CMECs. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05.

Fig. 3

Empagliflozin reduces I/R-induced mitochondrial fission, oxidative stress and apoptosis in CMECs. In vivo, mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts. The cells were cultured for 24 h and then used for functional analyses. (A–E) Proteins were isolated from CMECs, and Western blots were used to assess the expression of phosphorylated dynamin-related protein 1 (p-Drp1), Mff, mitochondrial fission 1 (Fis1), mitofusin 2 (Mfn2) and optic atrophy 1 (Opa1). (F–H) Immunofluorescence assay of the mitochondrial morphology. The average mitochondrial length was recorded to reflect mitochondrial fission. At least 100 mitochondria from 10 CMECs were used to evaluate the number of CMECs with fragmented mitochondria. (I–K) Mitochondrial and cytoplasmic ROS levels in CMECs were determined using immunofluorescence analyses. Mitochondrial ROS were assessed using MitoSOX™ Red, while cytoplasmic ROS were measured using CM-H2DCFDA. (L–N) The activity levels of intracellular anti-oxidative molecules such as GSH, SOD and GPX in CMECs were determined using commercially available ELISA kits. (O–P) JC-1 staining was used to assess the mitochondrial membrane potential in CMECs. A reduced red-to-green immunosignal of JC-1 indicates an abnormal mitochondrial membrane potential. (Q) mPTP opening in CMECs was determined based on the fluorescence intensity of TMRE. (R) An ELISA was used to assess capsase-9 levels in CMECs. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Empagliflozin activates FUNDC1-dependent mitophagy through the AMPKα1/ULK1 pathway.In vivo, mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts. The cells were cultured for 24 h and then used for functional analyses. (A–F) Proteins were isolated from reperfused heart tissues, and the levels of mitophagy-related proteins were analyzed through Western blots. (G–H) The mt-Kemia assay was used to analyze mitophagy in CMECs. (I–L) Proteins were isolated from CMECs, and the levels of p-AMPK, p-ULK1 and p-FUNDC1 were analyzed through Western blots. CC was administered to mice to inhibit empagliflozin-induced AMPK activation. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05.

Fig. 5

Ablation of FUNDC1 or AMPKa1 prevents empagliflozin from protecting against I/R-induced microvascular damage.In vivo, AMPKα1^EKO, FUNDC1^EKO or Tie2^Cre (control) mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. (A) An electron microscope was used to detect changes in the microvascular structure of the reperfused heart. Yellow arrows indicate microvascular endothelial cell inflation and luminal stenosis. (B–D) Proteins were isolated from reperfused heart tissues, and the levels of eNOS and ET-1 were analyzed through Western blots. (E, F) Immunohistochemistry assay of ICAM1, an endothelium-specific adhesive factor. (G, H)In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts and cultured for 24 h. Then, FITC-dextran clearance and TER assays were used to analyze the endothelial barrier function and integrity of the CMECs. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Empagliflozin sustains mitochondrial homeostasis in CMECs through the AMPKα1/ULK1/FUNDC1 pathway.In vivo, AMPKα1^EKO, FUNDC1^EKO or Tie2^Cre (control) mice were assigned to the sham operation group or the myocardial I/R injury group. Empagliflozin (10 mg/kg/d) was administered seven days before myocardial I/R injury. In vitro, CMECs were isolated from I/R- or empagliflozin-treated hearts. The cells were cultured for 24 h and then used for functional analyses. (A–C) Immunofluorescence assays were used to assess the mitochondrial morphology in CMECs. The average mitochondrial length was recorded to reflect mitochondrial fission. At least 100 mitochondria from 10 CMECs were used to evaluate the number of CMECs with fragmented mitochondria. (D–F) Mitochondrial and cytoplasmic ROS levels in CMECs were determined using immunofluorescence assays. Mitochondrial ROS were assessed using MitoSOX™ Red, while cytoplasmic ROS were measured using CM-H2DCFDA. (G–I) The activity levels of intracellular anti-oxidative molecules such as GSH, SOD and GPX in CMECs were determined using commercially available ELISA kits. (J–K) JC-1 staining was used to determine the mitochondrial membrane potential in CMECs. A reduced red-to-green immunosignal of JC-1 indicates an abnormal mitochondrial membrane potential. Experiments were repeated at least three times and the data are shown as mean ± SEM (n = 6 mice or three independent cell isolations per group). *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.