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Cited by 550Diabetes Treatment and ManagementCardiovascular Function and Risk FactorsMitochondrial Function and Pathology

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Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission

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Zhou, Hao, Wang, Shuyi, Zhu, Pingjun, Hu, Shunying, Chen, Yundai, Ren, Jun·2018·Redox Biology
DOI 10.1016/j.redox.2017.12.019PMC PMC5756062
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Abstract

Impaired cardiac microvascular function contributes to diabetic cardiovascular complications although effective therapy remains elusive. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor recently approved for treatment of type 2 diabetes, promotes glycosuria excretion and offers cardioprotective actions beyond its glucose-lowering effects. This study was designed to evaluate the effect of empagliflozin on cardiac microvascular injury in diabetes and the underlying mechanism involved with a focus on mitochondria. Our data revealed that empagliflozin improved diabetic myocardial structure and function, preserved cardiac microvascular barrier function and integrity, sustained eNOS phosphorylation and endothelium-dependent relaxation, as well as improved microvessel density and perfusion. Further study suggested that empagliflozin exerted its effects through inhibition of mitochondrial fission in an adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner. Empagliflozin restored AMP-to-ATP ratio to trigger AMPK activation, suppressed Drp1 S616 phosphorylation, and increased Drp1 S637 phosphorylation, ultimately leading to inhibition of mitochondrial fission. The empagliflozin-induced inhibition of mitochondrial fission preserved cardiac microvascular endothelial cell (CMEC) barrier function through suppressed mitochondrial reactive oxygen species (mtROS) production and subsequently oxidative stress to impede CMEC senescence. Empagliflozin-induced fission loss also favored angiogenesis by promoting CMEC migration through amelioration of F-actin depolymerization. Taken together, these results indicated the therapeutic promises of empagliflozin in the treatment of pathological microvascular changes in diabetes.

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Figures

Fig. 1 Effect of empagliflozin treatment on cardiac function and morphology in diabetes: Experimental diabetes was induced with STZ and mice were treated with empagliflozin (10 mg/kg/d) for 20 weeks (n = 6/group). A. Representative M-mode echocardiographic images collected using the parasternal long-axis view in each group; B–E. Quantitative analysis of cardiac function performed using echocardiography; F–G. Cardiac fibrosis following empagliflozin treatment assessed using Masson's staining; H. Representative electron microscopy images of cardiomyocytes in each group. Diabetes led to myocyte dissolution, muscular fiber twists and Z line disappearance, the effects of which were reversed by empagliflozin. Diabetes caused mitochondrial swelling and crista fragmentation (yellow arrow), which were in contrast with the clearly visible and regular mitochondria seen in controls. Empagliflozin recovered diabetes-distorted mitochondrial structural integrity. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.
Fig. 1 Effect of empagliflozin treatment on cardiac function and morphology in diabetes: Experimental diabetes was induced with STZ and mice were treated with empagliflozin (10 mg/kg/d) for 20 weeks (n = 6/group). A. Representative M-mode echocardiographic images collected using the parasternal long-axis view in each group; B–E. Quantitative analysis of cardiac function performed using echocardiography; F–G. Cardiac fibrosis following empagliflozin treatment assessed using Masson's staining; H. Representative electron microscopy images of cardiomyocytes in each group. Diabetes led to myocyte dissolution, muscular fiber twists and Z line disappearance, the effects of which were reversed by empagliflozin. Diabetes caused mitochondrial swelling and crista fragmentation (yellow arrow), which were in contrast with the clearly visible and regular mitochondria seen in controls. Empagliflozin recovered diabetes-distorted mitochondrial structural integrity. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.CC BY-NC-ND · PubMed Central
Fig. 2 Empagliflozin improves myocardial microcirculation perfusion by increasing microvessel density and reducing vascular remodeling. A. Representative MCE images were taken using a constant infusion of microbubbles at 20 mL/min. The signal intensity was determined by capturing a 10-s high-energy sequence at a frame rate of 30 Hz;.B. A quantitative analysis of perfusion was performed using Research-Arena software (Tomtech, Germany). A: plateau intensity, β: flow velocity. Myocardial blood flow (A × β) profiles in the basal septum for different groups (n = 6/group); C–D. Frozen cardiac sections were incubated with CD31 antibody to assess microvascular numbers by performing fluorescence microscopy; E. Microvascular image detection via ink staining; F–G. Immunohistochemistry of p-eNOS (Ser1177) expression; H–I. Endothelial-dependent and endothelial-independent relaxation responses were assessed by applying Ach (10−9–10−5 M) or SNP (10−10–10−6 M). J. Histological analysis of vascular fibrosis with Masson's staining; K–L. TEM was performed to observe changes in the vascular basement membrane. Diabetes induced hyperplasia of the vascular basement, which was reversed by empagliflozin. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.
Fig. 2 Empagliflozin improves myocardial microcirculation perfusion by increasing microvessel density and reducing vascular remodeling. A. Representative MCE images were taken using a constant infusion of microbubbles at 20 mL/min. The signal intensity was determined by capturing a 10-s high-energy sequence at a frame rate of 30 Hz;.B. A quantitative analysis of perfusion was performed using Research-Arena software (Tomtech, Germany). A: plateau intensity, β: flow velocity. Myocardial blood flow (A × β) profiles in the basal septum for different groups (n = 6/group); C–D. Frozen cardiac sections were incubated with CD31 antibody to assess microvascular numbers by performing fluorescence microscopy; E. Microvascular image detection via ink staining; F–G. Immunohistochemistry of p-eNOS (Ser1177) expression; H–I. Endothelial-dependent and endothelial-independent relaxation responses were assessed by applying Ach (10−9–10−5 M) or SNP (10−10–10−6 M). J. Histological analysis of vascular fibrosis with Masson's staining; K–L. TEM was performed to observe changes in the vascular basement membrane. Diabetes induced hyperplasia of the vascular basement, which was reversed by empagliflozin. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.CC BY-NC-ND · PubMed Central
Fig. 3 Empagliflozin maintains microvessel integrity and barrier function. A–B. The co-immunofluorescence of VE-cadherin and CD31 indicates the continuity of gap junctional proteins; C–D. Representative fluorescence images for intercellular adhesion molecule (ICAM)-1; E–F. Immunohistochemical staining of vascular cell adhesion molecule (VCAM)-1; G. HE staining to assess red blood cell adhesion to microvessel walls in different groups; H–I. A TUNEL assay was performed to assess CMEC apoptosis. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.
Fig. 3 Empagliflozin maintains microvessel integrity and barrier function. A–B. The co-immunofluorescence of VE-cadherin and CD31 indicates the continuity of gap junctional proteins; C–D. Representative fluorescence images for intercellular adhesion molecule (ICAM)-1; E–F. Immunohistochemical staining of vascular cell adhesion molecule (VCAM)-1; G. HE staining to assess red blood cell adhesion to microvessel walls in different groups; H–I. A TUNEL assay was performed to assess CMEC apoptosis. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group.CC BY-NC-ND · PubMed Central
Fig. 4 Empagliflozin diminishes diabetic-induced mitochondrial fragmentation and regulated the balance of proteins responsible for mitochondrial fission and fusion. CMECs were obtained from the control group, diabetic group and empagliflozin group. After isolation, empagliflozin-treated CMECs were treated with FCCP to trigger mitochondrial fission. A. EM was performed to observe mitochondrial fission in vivo. Diabetes induced mitochondrial disintegration into numerous round fragments of varying sizes accompanied by the disappearance of crista (white arrow), unlike the characteristic reticulo-tubular mitochondrial morphology seen in the control group, and these changes were reversed by empagliflozin; B. CD31 immunocytochemistry of CMECs and Dil-acetylated low-density lipoprotein intake assay. (green, CD31; red, Dil-LDL); C. CMECs were isolated and cultured in vitro, and MitoTracker Deep Red was used to label the mitochondria, whose morphology was analyzed using fluorescence microscopy; D. Co-localization of Drp1 and mitochondria. The boxed area under each micrograph represents the amplification of the white square. More Drp1 was located on fragmented mitochondria, while empagliflozin reduced Drp1 migration onto mitochondria; E. Changes in proteins related to mitochondrial fission and fusion. Empagliflozin reduced fission-associated factors and increased fusion-involved protein levels. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group.
Fig. 4 Empagliflozin diminishes diabetic-induced mitochondrial fragmentation and regulated the balance of proteins responsible for mitochondrial fission and fusion. CMECs were obtained from the control group, diabetic group and empagliflozin group. After isolation, empagliflozin-treated CMECs were treated with FCCP to trigger mitochondrial fission. A. EM was performed to observe mitochondrial fission in vivo. Diabetes induced mitochondrial disintegration into numerous round fragments of varying sizes accompanied by the disappearance of crista (white arrow), unlike the characteristic reticulo-tubular mitochondrial morphology seen in the control group, and these changes were reversed by empagliflozin; B. CD31 immunocytochemistry of CMECs and Dil-acetylated low-density lipoprotein intake assay. (green, CD31; red, Dil-LDL); C. CMECs were isolated and cultured in vitro, and MitoTracker Deep Red was used to label the mitochondria, whose morphology was analyzed using fluorescence microscopy; D. Co-localization of Drp1 and mitochondria. The boxed area under each micrograph represents the amplification of the white square. More Drp1 was located on fragmented mitochondria, while empagliflozin reduced Drp1 migration onto mitochondria; E. Changes in proteins related to mitochondrial fission and fusion. Empagliflozin reduced fission-associated factors and increased fusion-involved protein levels. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group.CC BY-NC-ND · PubMed Central
Fig. 5 Empagliflozin protects CMECs against cellular senescence and barrier dysfunction by suppressing fission-mediated mtROS overproduction. A. Representative images of β-galactosidase staining, which was the marker for aged cells. B. Quantification of β-galactosidase-positive endothelial cells. C. The immunofluorescence of intracellular ROS and mtROS as assessed by DCF-DA and MitoSOX™ Red Mitochondrial Superoxide Indicator. Mitoquinone (MitoQ, 2 μM) was applied for 24 h to reduce the cellular oxidative damage. D–E. Cell cycle distribution was detected by performing flow cytometric quantification. Loss of fission due to empagliflozin and mdivi1 contributed to the cell cycle transition from G0/G1 to S phase, suggesting the anti-senescence effects of empagliflozin occur via fission inhibition. F. A TER assay was performed to detect CMEC barrier function during mitochondrial fission. TER increases when endothelial cells adhere and spread out and decreases when endothelial cells retract or lose adhesion, reflecting endothelial barrier integrity. G. FITC-dextran clearance was measured to assess changes in endothelial permeability. FITC-dextran was applied on top of the inserts and allowed to permeate through cell monolayers. The increased endothelial permeability resulted in the retention of more FITC-dextran. Thus, FITC content remaining in the plate indicates the extent of CMEC permeability. H–J. The change of expression of p-eNOS (Ser1177), ICAM1 and VCAM1. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group, %P < 0.05 vs. Diabetic + EMPA + FCCP group.
Fig. 5 Empagliflozin protects CMECs against cellular senescence and barrier dysfunction by suppressing fission-mediated mtROS overproduction. A. Representative images of β-galactosidase staining, which was the marker for aged cells. B. Quantification of β-galactosidase-positive endothelial cells. C. The immunofluorescence of intracellular ROS and mtROS as assessed by DCF-DA and MitoSOX™ Red Mitochondrial Superoxide Indicator. Mitoquinone (MitoQ, 2 μM) was applied for 24 h to reduce the cellular oxidative damage. D–E. Cell cycle distribution was detected by performing flow cytometric quantification. Loss of fission due to empagliflozin and mdivi1 contributed to the cell cycle transition from G0/G1 to S phase, suggesting the anti-senescence effects of empagliflozin occur via fission inhibition. F. A TER assay was performed to detect CMEC barrier function during mitochondrial fission. TER increases when endothelial cells adhere and spread out and decreases when endothelial cells retract or lose adhesion, reflecting endothelial barrier integrity. G. FITC-dextran clearance was measured to assess changes in endothelial permeability. FITC-dextran was applied on top of the inserts and allowed to permeate through cell monolayers. The increased endothelial permeability resulted in the retention of more FITC-dextran. Thus, FITC content remaining in the plate indicates the extent of CMEC permeability. H–J. The change of expression of p-eNOS (Ser1177), ICAM1 and VCAM1. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group, %P < 0.05 vs. Diabetic + EMPA + FCCP group.CC BY-NC-ND · PubMed Central
Fig. 6 Suppression of fission by empagliflozin contributes to CMEC migration and neovascularization by preserving F-actin homeostasis. A–B. The endothelial migration response to SDF-1 was analyzed by performing a transwell assay. C. Co-immunofluorescence of F-actin and mitochondria to establish the role of mitochondrial fission in F-actin homeostasis. Fragmented mitochondria were accompanied by F-actin fiber disorder or dissolution, which was reversed by empagliflozin or mdivi1. D–F. Changes in the expression of F-actin and its metabolite G-actin as assessed by western blotting. G–H. A wound-healing assay was carried out to detect cell motility with F-actin depolymerization. I–J. The influence of mitochondrial fission on the capacity of CMECs to undergo angiogenesis via F-actin was measured by performing a Matrigel assay in vitro. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group.
Fig. 6 Suppression of fission by empagliflozin contributes to CMEC migration and neovascularization by preserving F-actin homeostasis. A–B. The endothelial migration response to SDF-1 was analyzed by performing a transwell assay. C. Co-immunofluorescence of F-actin and mitochondria to establish the role of mitochondrial fission in F-actin homeostasis. Fragmented mitochondria were accompanied by F-actin fiber disorder or dissolution, which was reversed by empagliflozin or mdivi1. D–F. Changes in the expression of F-actin and its metabolite G-actin as assessed by western blotting. G–H. A wound-healing assay was carried out to detect cell motility with F-actin depolymerization. I–J. The influence of mitochondrial fission on the capacity of CMECs to undergo angiogenesis via F-actin was measured by performing a Matrigel assay in vitro. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic + EMPA group.CC BY-NC-ND · PubMed Central
Fig. 7 AMPK signaling is activated by empagliflozin via increases in the AMP/ATP ratio, contributing to Drp1 modifications and subsequently mitochondrial fission. A–D. Empagliflozin increased p-AMPK levels. Activated AMPK was involved in regulating Drp1 phosphorylation. E. Empagliflozin increased the AMP/ATP ratio, which was responsible for AMPK activation. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic+EMPA group. AI: AICAR, cC: compound C.
Fig. 7 AMPK signaling is activated by empagliflozin via increases in the AMP/ATP ratio, contributing to Drp1 modifications and subsequently mitochondrial fission. A–D. Empagliflozin increased p-AMPK levels. Activated AMPK was involved in regulating Drp1 phosphorylation. E. Empagliflozin increased the AMP/ATP ratio, which was responsible for AMPK activation. Mean ± SD, *P < 0.05 vs. Control (Cont) group; #P < 0.05 vs. Diabetic group, &P < 0.05 vs. Diabetic+EMPA group. AI: AICAR, cC: compound C.CC BY-NC-ND · PubMed Central
Fig. 8 Scheme depicting proposed mechanisms involved in empagliflozin-offered protection against microvasculature damage in diabetes. Empagliflozin activates AMPK pathways through regulation of the AMP/ATP ratio. Activated AMPK pathways regulates Drp1 posttranscriptional phosphorylation modifications at Ser616 and Ser637, leading to the inability of Drp1 to translocate onto mitochondria and mitochondrial fission impairment. The loss of mitochondrial fission retards cellular senescence and preserves endothelial barrier/permeability by suppressing superfluous ROS. In consequence, endothelial migration and vascularization are improved by balanced F-actin degradation. Moreover, empagliflozin reduces CMEC apoptosis, increases cardiac microvessel density, promotes eNOS phosphorylation and alleviates vascular collagen deposition, leading to improved endothelial function and preserved vascular remodeling, ultimately lower levels of inflammatory cell penetration and better vascular relaxation. Through these aforementioned mechanisms, empagliflozin eventually facilitates diabetic myocardial perfusion and protects the heart against hyperglycemic injury.
Fig. 8 Scheme depicting proposed mechanisms involved in empagliflozin-offered protection against microvasculature damage in diabetes. Empagliflozin activates AMPK pathways through regulation of the AMP/ATP ratio. Activated AMPK pathways regulates Drp1 posttranscriptional phosphorylation modifications at Ser616 and Ser637, leading to the inability of Drp1 to translocate onto mitochondria and mitochondrial fission impairment. The loss of mitochondrial fission retards cellular senescence and preserves endothelial barrier/permeability by suppressing superfluous ROS. In consequence, endothelial migration and vascularization are improved by balanced F-actin degradation. Moreover, empagliflozin reduces CMEC apoptosis, increases cardiac microvessel density, promotes eNOS phosphorylation and alleviates vascular collagen deposition, leading to improved endothelial function and preserved vascular remodeling, ultimately lower levels of inflammatory cell penetration and better vascular relaxation. Through these aforementioned mechanisms, empagliflozin eventually facilitates diabetic myocardial perfusion and protects the heart against hyperglycemic injury.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Zhou, H., Wang, S., Zhu, P., Hu, S., Chen, Y., & Ren, J.
Affiliations
  • Chinese PLA General Hospital (CN)
  • University of Wyoming (US)
Corresponding author
  • Zhou, Hao
  • Chen, Yundai
  • Ren, Jun
ORCID
Venue
Redox Biology
Publisher
Elsevier BV
Published
2018-05
Volume
15
Pages
335-346
ISSN
2213-2317 (ISSN-L)
DOI
10.1016/j.redox.2017.12.019
PMC
PMC5756062
OpenAlex
W2777514005
Cited by
550
References
63
Citations by year
2026: 34 · 2025: 53 · 2024: 87 · 2023: 71 · 2022: 71 · 2021: 65 · 2020: 63 · 2019: 53 · 2018: 50
FWCI
35.91
Type
journal article
Language
en
Update notices
erratum (2024-05-01)
Open access
Licence
Locations
  • Redox Biology (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Natural Science Foundation of China (81441008, 81030002, 81102079, 81270186)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diabetes Treatment and Management
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiovascular Function and Risk Factors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
empagliflozin, SGLT2 inhibitors, diabetic cardiomyopathy, mitochondrial fission, AMPK, Drp1 phosphorylation, endothelial barrier function, endothelial cell senescence, mitochondrial reactive oxygen species, oxidative stress, angiogenesis, endothelial cell migration, F-actin depolymerization, eNOS phosphorylation, type 2 diabetes
MeSH terms
AMP-Activated Protein Kinase Kinases, Animals, Benzhydryl Compounds, Diabetes Mellitus, Type 2, Glucosides, Heart Injuries, Humans, Mitochondria, Protein Kinases, Endothelial Cells, Mice, Mitochondrial Dynamics

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