TPH104m

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

A B S T R A C T
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 micro- vascular 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 micro- vascular 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, sup- pressed Drp1S616 phosphorylation, and increased Drp1S637 phosphorylation, ultimately leading to inhibition of mitochondrial fission. The empagliflozin-induced inhibition of mitochondrial fission preserved cardiac micro- vascular 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 depoly- merization. Taken together, these results indicated the therapeutic promises of empagliflozin in the treatment of pathological microvascular changes in diabetes.

1.Introduction
CThe cardiac microvasculature, which primarily consists of cardiac microvascular endothelial cell (CMEC) located at the circulatory ter- minus, governs myocardial perfusion and coronary reserve [1]. Given the direct contact between the microvasculature and blood flow, CMECs are more vulnerable to hyperglycemic damage as opposed to cardiomyocytes. With the onset and development of diabetes mellitus, impaired CMEC viability and cell migration occur and contribute to the compromised endothelial regulation of vascular homeostasis, favoring a pro-inflammatory state that ultimately results in vascular rarefaction and diabetic vasculopathy [2]. In consequence, dampened myocardial perfusion and cardiac ischemia develop due to a shortage of vascular supply to cope with the cardiac demand, predisposing diabetic patients to cardiovascular complications [3]. Therefore, the hunt for means toprotect the cardiac microvasculature against hyperglycemic damage is essential to retard or alleviate diabetic macrovascular complications [4].Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, was recently developed as an anti-diabetic agent to promote urinal glucose excretion or glycosuria in an insulin-independent manner [5,6]. Empagliflozin may also lower cardiovascular mortality, all-cause mor- tality and hospitalization for heart failure by 38%, 35% and 32%, re- spectively, over a median duration of 3.1 years [7]. In addition to the improved glycemic parameters, empagliflozin helps to switch body metabolism towards lipid utilization and reduces systolic blood pres- sure in the absence of tachycardia [8], thus favoring a cardioprotective property of the SGLT2 inhibitor [9].

Nonetheless, the impact of em- pagliflozin in diabetic microvascular complications, particularly car- diac microvascular damage, remains largely unknown.The mitochondrial content is modest in endothelial cells compared to other cell types with a higher energy requirement, indicating a pri- mary role for mitochondria as essential signaling organelles in vascular endothelium [10,11]. Abnormal mitochondrial dynamics, in particular mitochondrial fission, has been reported to play an important role in the pathogenesis of diabetic nephropathy microvascular complications[12] through mechanism in part associated with transmission of glu- cose toxic signals [13]. Nonetheless, whether empagliflozin treatment alleviates cardiac microvascular injury in particular by way of mi- tochondrial fission remains unknown. As a result of excessive mi- tochondrial fission, cristae disorganization, membrane permeabiliza- tion and release of pro-apoptotic proteins [14,15] develop and contribute to cellular death, en route to the ultimate cardiac-renal in- jury in hyperglycemia [12]. In addition to cell death, blunted migration and cellular senescence of endothelial cells are also involved in diabetic microvascular damage. However, little is known with regards to the role of mitochondrial fission in CMEC migration and senescence.

Dynamin-related protein 1 (Drp1) serves as a critical effector of mitochondrial fission given the redistribution of GFP-tagged Drp1 from a predominant cytosolic location to predicted sites of division along mitochondrial tubules [16,17]. Drp1 phosphorylation is a permissive step to facilitate the shift of Drp1 onto mitochondria, a process regu- lated by post-translational events including phosphorylation, sumoy- lation, ubiquitination, and nitrosylation [18,19]. Several upstream signaling molecules were reported to promote Drp1 activation, in- cluding Rho-associated coiled-coil containing protein kinase (ROCK1) and AMPK. ROCK triggers fission by phosphorylating Drp1 at the Serine616 (Ser616) residue in kidney podocytes and endothelial cells [12]. In contrast, Drp1 is inactivated via phosphorylation at Ser637 in response to AMPK activation in aortic endothelial cells [20]. These data support a pivotal role for phosphorylation in the regulation of Drp1 and its translocation onto mitochondrial membranes. However, whether empagliflozin affects diabetic microvascular complications and mi- tochondrial quality through Drp1 modification remains largely un- explored. Here, our data suggested beneficial response of empagliflozin on cardiac microvascular structure and function, revealing an im- portant role for the SGLT2 inhibitor in mitochondrial fission and CMEC viability, migration and proliferation.

2.Methods
All experimental procedures described here were in accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animal and were approved by the PLA General Hospital Institutional Animal Care and Use Committee. In brief, 8-week-old C57BL/6J wild-type (WT) mice were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) for 5 consecutive days. After the first 4 weeks of diabetes (confirmed by blood glucose levels > 16 mmol/L after a six-hour daytime fasting), mice (12-week-old) were treated with empagliflozin (10 mg/kg/d, defined as the empagliflozin group) or the mitochondrial fission inhibitor mdivi-1 (1.2 mg/kg/d, designed as the mdivi1 group) for 20 weeks. At the end of treatment, all mice (32-week- old) were euthanized, and hearts were collected for further experi- mentations. Empagliflozin was provided by the Boehringer Ingelheim Pharma GmbH & Co. KG, Germany, and was administered via oral gavage (0.5% hydroxyethylcellulose was used as the vehicle).Echocardiography was performed using echocardiogram (14.0 MHz, Sequoia C512; Acuson, Germany) to monitor changes in cardiac function [14]. Myocardial contrast echocardiography (MCE) was performed using a 14 MHz linear transducer (Acuson Sequoia C512 system) with the constant infusion of microbubbles [10% Perflutrenlipid microspheres (Definity, Lantheus Imaging) diluted tenfold in sterile saline] at 20 mL/min as previously described by our group [1].Gelatin-ink perfusion was carried out using ink warmed to 37 °C along with 3% gelatin (gelatin-ink staining via jugular vein perfusion).

Room temperature was maintained at 25–30 °C. When the limbs turned black, the great vessels of the cardiac base and the superior and inferior vena cava were separated and were ligated. The hearts were subse- quently maintained at 4 °C for at least 1 h, prior to heart removal and fixation in 4% paraformaldehyde and processed for cryosectioning [21].CMECs were isolated from the hearts of normal, diabetic, empagli- flozin-treated diabetic mice and diabetic mice treated with Mdivi1 (a mitochondrial fission inhibitor) using the enzyme dissociation method described previously by our group [22]. The purity of cultured cells was assessed by performing CD31 staining and the uptake of acetylated low- density lipoprotein. To induce mitochondrial fission, CMECs obtainedfrom empagliflozin-treated mice were treated with 5 μM FCCP for 2 h.Reactive oxygen species (ROS), an indicator of oxidant status, are involved in the injury of CMECs in the setting of diabetic oxidative stress. Both DCF-DA (10 μM) (Invitrogen, Germany) and MitoSOX™ Red Mitochondrial Superoxide Indicator (Molecular Probes, USA) were used to monitor the change of intracellular ROS and mitochondrial ROS(mtROS), respectively, using a laser confocal microscope (Olympus) [23].For cell cycle analysis, CMECs were isolated from the heart and were resuspended in phosphate-buffered saline (PBS) followed by fixation with ice-cold 70% ethanol for 24 h. The fixed cells were rinsed and then resuspended in 50 μg/mL propidium iodide for 30 min in the dark [24]. Flow cytometric analysis was performed using a BD FACS-Calibur cytometer as described previously [25].The migration assay of CMECs was evaluated using 24-well trans- well chambers (Coring, USA).

First, 1 × 105 CMECs were seeded in the upper chamber containing serum-free media. The chemotactic agent SDF-1 (100 ng/mL, Sigma-Aldrich, USA) was added to the lower chamber to induce CMEC migration [26]. After a 12-h incubation at 37 °C, non-migrating cells in the upper chamber were carefully re- moved using a cotton swab, cells that traversed through the membrane were fixed in methanol, stained with 0.05% crystal violet [27].As to the wound healing assay, 1 × 106 CMECs were seeded onto 36-mm plates to reach 80–90% confluency. An artificial wound was performed using P200 pipette tip scratching on the confluent cell monolayer [28]. Photomicrograph was taken immediately (time 0 h), then cells were incubated in DMEM containing 1% fetal bovine serum. The mobilization of cells and closing of scratch wound were observed 24 h later.A FITC-dextran clearance assay was performed to monitor changes in CMEC permeability. Cells were incubated with FITC-dextran (finalconcentration: 1 mg/mL), and were allowed to permeate through the cell monolayer. Two hours later, the FITC content remaining in the plate was measured using a fluorescent plate reader (Bio-Rad, USA) to detect the extent of permeability. TER is a measure of ionic con- ductance of endothelial cells and is used to assess junctional function. TER decreases when endothelial cells retract or lose adhesion. Using an in vitro Vascular Permeability Assay Kit (ECM640, Millipore, USA), CMECs were seeded onto collagen-coated inserts at a density of 100,000 cells/insert.

After reaching confluence, an electrical endothelial re- sistance system (Millipore, USA) was used to measure TER per our previous description [1].The in vitro endothelial tube formation assay was performed as previously described [29]. Briefly, 100 μL of Matrigel (BD Bioscience) was added to each well of a 24-well plate and allowed to polymerize at 37 °C for 30 min [30]. CMECs were suspended in FBS-free DMEM medium and seeded in each well at a concentration of 1 × 105 cells/well. Cells were incubated at 37 °C. Each treatment was repeated in triplicates [31]. After 6 h, cells were examined under a light microscope to assess the formation of capillary-like structures. The branch points of the formed tubes, which represent the degree of angiogenesis in vitro, were scanned and quantified in five low-power fields (200×).Thoracic aorta was immediately dissected and immersed in chilled Krebs–Henseleit (K-H) solution and bubbled with 95% O2 and 5% CO2 (pH 7.4). The descending aortic rings were pre-contracted with U46619 (30 nM). At the plateau of contraction, endothelium-dependent and-independent vasodilation were determined using ACh (10−9–10−5 M) and SNP (10−10–10−6 M), respectively, at the given concentration after 15-min incubation in the bath.Myocardial tissues were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin followed by dehydration in graded ethanol solutions and in toluene. Five-micron-thick sections were stained with hematoxylin and eosin (H.E.) or Masson’s stain and examined via light microscopy for histopathological analysis. Microvascular density was observed using a CD31 antibody (1:1500, Abcam), eNOS phosphor- ylation (Ser1177) (1:1000, Abacm) and endothelial barrier integrity was assessed with VE-cadherin (1:1000, Abcam). For immunofluorescence staining in vitro, cells were fixed with 4% paraformaldehyde, permea- bilized with 0.3% Triton X-100, and blocked with 10% goat serum al- bumin (Invitrogen, USA.).

Specimens were subsequently incubated with primary antibodies against Drp1 (1:1000, Abcam) and F-actin (1:1000, Abcam). DAPI staining (Sigma-Aldrich, USA) and mitochondrion-se- lective MitoFluor™ staining (Molecular Probes, USA) were performed to label nuclei and mitochondria, respectively [32].Proteins (60–80 µg) were loaded on a 10–15% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were trans- ferred to a PVDF membrane. Membrane were blocked with 5% nonfat dried milk (in TBST) for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies. The membrane was subse- quently washed with TBST (5 min × 3) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature [33]. After washing with TBST (5 min × 3), bands were detected by enhanced chemilumines- cence substrate (Applygen). The antibodies used in the present were asfollows: Drp1 (1:1000, Abcam, #ab56788), p-Drp1 (Ser616) (1:1000, Cell Signaling Technology, #3455), p-Drp1 (Ser637) (1:1000, Abcam, #ab193216), Fis1 (1:1000, Abcam, #ab71498), Mfn2 (1:1000, Abcam, #ab56889), Mfn1 (1:1000, Abcam, #ab57602), Mff (1:1000, Cell Sig-naling Technology, #86668), F-actin (1:1000, Abcam, #ab205), G- actin (1:1000, Abcam, #ab200046), p-eNOS (Ser1117) (1:1000, Abcam, #ab184154), ICAM1 (1:1000, Abcam, #ab119871), VCAM1 (1:1000, Abcam, #ab134047), AMPK (1:1000, Abcam, #ab131512), p- AMPK (1:1000, Abcam, #ab23875) [34].Tissues were fixed in 2.5% glutaraldehyde at 4 °C. After fixation in 1% osmium tetraoxide, the tissues were dehydrated and then embedded in araldite CY 618. Slides (50 nm) were stained with lead citrate acid and uranium acetate to observe cardiac microvasculature through a Hitachi H600 Electron Microscope (Hitachi, Japan) [24]. Images were at 15,000× magnification.Data were presented as mean ± standard deviation (SD) from at least three independent experiments and were analyzed using one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05. 3.Results Echocardiography was performed to evaluate changes in cardiac function. Compared with the control group, diabetic mice exhibited overtly dampened diastolic function as manifested by a decline in the E/A ratio and an increased left ventricular volume in systole (LV vol-s), the effect of which was reversed by empagliflozin (Fig. 1A–E). These functional changes were attributable to alterations in cardiac structure. Histological analysis using Masson Trichrome's staining revealed sig- nificantly elevated collagen fiber deposition in diabetes, the effect of which was negated by empagliflozin (Fig. 1F–G). In addition, electron microscopy (EM) evaluation revealed overt cardiomyocyte dissolution, muscular fiber twisting and Z line disappearance in diabetes, the effects of which were greatly attenuated by empagliflozin treatment (Fig. 1H). Furthermore, empagliflozin treatment helped to maintain mitochon- drial integrity in diabetes as evidenced by the appearance of frag- mented mitochondria characterized by crista deformation (yellow ar- rows presented in Fig. 1H). These data favor a cardioprotective property of empagliflozin on diabetic myocardial structural and func- tional damages. Furthermore, the effects of empagliflozin on hemody- namics and body weight were evaluated. Diabetic mice exhibited a lower body weight gain and higher levels of systolic blood pressure (SBP) and blood glucose. Not surprisingly, empagliflozin improved body weight gain and decreased the levels of SBP and blood glucose possibly by promoting glycosuria (Table 1). Myocardial function is dependent upon continuous blood flow governed by microcirculation, which is susceptible to hyperglycemic damage. MCE was performed to evaluate myocardial microcirculatory perfusion. Compared to the control group, diabetic mice exhibited a decrease in microvascular perfusion as shown by scattered perfusion defects (Fig. 2A–B). However, mice in empagliflozin-treated diabetic group developed much smaller zones of perfusion defect. Similar results were obtained in mdivi1-treated diabetic mice. To discern the underlying mechanism, fundamental micro- circulatory perfusion factors including microvascular density, vessel stenosis, eNOS phosphorylation (Ser1177) and endothelial relaxation function, were evaluated. As illustrated in Fig. 2C–D, diabetes triggered a drop in the number of CD31+ microvessels, similar to the vascular ink staining depicting the sparse of vascular beds (Fig. 2E), the effects of which were reversed by empagliflozin. Moreover, diabetes-induced decrease in eNOS phosphorylation (Ser1177) was reinstated by empa- gliflozin (Fig. 2F–G). Changes in eNOS phosphorylation in the micro- vasculature in response to diabetes and empagliflozin were echoed by the responses in endothelial relaxation in isolated aorta (Fig. 2H–I). In accordance with the functional alterations, diabetes-induced changes in microvascular morphology including fibrosis (Fig. 2J) and basement membrane thickening (Fig. 2K–L) (assessed by Masson's staining and TEM, respectively) were largely ameliorated by empagliflozin, sug- gesting a role for empagliflozin to retard remodeling in diabetic vas- culopathy. Notably, treatment with the mitochondrial fission inhibitor mdivi1 in diabetic mice also increased microvessel density and lessened vascular remodeling. Damage to microvessel integrity and barrier function is considered the initial step in vascular complications in diabetes. VE-cadherins, junctional proteins expressed on endothelial cells, are vital players for endothelial integrity and vascular permeability. Our data revealed that diabetes significantly downregulated VE-cadherin fluorescence in the microvasculature, the effect of which was nullified by empagliflozin (Fig. 3A–B). In addition, diabetes significantly upregulated levels of adhesive proteins including ICAM1 and VCAM1, the effects of which were reversed by empagliflozin (Fig. 3C–F). These diabetes-induced effects should impact the accumulation of erythrocytes in the micro- vessels due to the stop of turbulent blood flow or the secondary effect owing to the capillary blockage by other cells such as leukocytes. However, empagliflozin considerably retarded such changes (Fig. 3G). These data collectively revealed the beneficial effects of empagliflozin on the microvessel integrity and barrier function in diabetes, favoring a significant decrease for the risk for vascular inflammation and potential microthrombus formation. Similar results were obtained in mdivi1- treated group. Furthermore, the collapse of microvessel integrity and barrier function may be the result of CMEC dysfunction or death given the greater numbers of TUNEL+ cells appeared in diabetic hearts. In- terestingly, empagliflozin or mdivi1 prevented diabetes-induced rises in the percentage of TUNEL+ cells (Fig. 3H–I), supporting the pro-survival capacity of empagliflozin on hyperglycemia-mediated CMEC apoptosis. To determine the possible mechanism(s) of action through which empagliflozin protects the microvasculature against diabetic injury, mitochondrial fission was monitored. First, EM was performed to observe mitochondrial fission in the microvasculature in vivo. Our re- sult noted that mitochondria became smaller and punctate with sig- nificantly shorter lengths in diabetic microvasculature compared with the control group (white arrows in Fig. 4A). However, the majority of mitochondria in empagliflozin-treated group exhibited a long fila- mentous morphology, indicating the likelihood ability of empagliflozin to suppress diabetes-induced mitochondrial fission (Fig. 4A). Further- more, CMECs were isolated from normal, diabetic and empagliflozin- treated diabetic mice to assess mitochondrial fission in vitro. CMECs from diabetic mice treated with Mdivi1 were used as the negative controls. Isolated CMECs were identified by CD31 positivity and by performing a DiL-Ac-LDL phagocytic test (Fig. 4B). As shown in Fig. 4C, the mitochondria in diabetic cells had fewer free ends compared with those in control group, whereas the application of mdivi1 or empagli- flozin reduced the numbers of mitochondrial fragments. Numerous bulb-like structures were observed at the base of mitochondrial tubules in empagliflozin-treated cells but not in diabetic cells. However, treatment with the fission activator FCCP (5 μM for 2 h) cancelled off empagliflozin-offered protective effect on fission. Moreover, Drp1 shut- tling to the mitochondrial surface is an indispensable process for fission, and the subcellular localization of Drp1 was observed in empagliflozin group. Diabetes increased Drp1 migration onto the fragmented mitochondria carrying greater amounts of free debris (Fig. 4D). How- ever, Drp1 foci on the mitochondria were clearly decreased in CMECs treated with empagliflozin or mdivi1. Notably, FCCP suppressed the protective action of empagliflozin on fission, leading to a greater number of mitochondrial fragments marked with Drp1. These data strongly suggest the ability of empagliflozin to repress excessive mi- tochondrial fission triggered by long-term diabetes in CMECs. Indeed, mitochondrial fission was manipulated by the balance between fission- and fusion-related proteins. Diabetes significantly diminished the levels of fusion-related proteins, including Mfn1 and Mfn2. However, empa- gliflozin effectively up-regulated the fusion-related proteins while down-regulating the fission-associated factors such as Fis1 and Mff (Fig. 4E). To gain more insight into the role of mitochondrial fission in diabetic microvascular injury, we assessed changes in CMEC senescence by performing β-galactosidase staining. Diabetes evoked a higher percen- tage of β-gal+ cells compared with that in the control groups (Fig. 5A–B). However, inhibition of fission in diabetic CMECs via the application of mdivi1 reduced the percentage of β-gal+ cells, re- miniscent of empagliflozin treatment. In contrast, induction of fission resulted in the rebound of β-gal+ cells in the empagliflozin group, in- dicating empagliflozin inhibited cellular senescence by suppressing fission (Fig. 5A–B). Oxidative stress serves as the primary cue underlying hyperglycemic cellular senescence, whereas mitochondria function as an important source of ROS. To confirm whether fission aggravated senescence through oxidative stress, an ROS probe was applied. Diabetic CMECs produced higher levels of mtROS and intracellular ROS (Fig. 5C), while mdivi1 and empagliflozin treatment restricted this production. Inter- estingly, fission activation offset the ROS-clearing effects of empagli- flozin, indicating that a permissive role of fission in oxidative stress induction. Excessive ROS hampered the cell cycle transition from G0/ G1 to S phase, which was reversed by the application of an mtROS scavenger, empagliflozin and mdivi1 (Fig. 5D–E). Moreover, preventing cellular senescence by neutralizing fission-induced ROS also reduced the residual FITC-dextran content and increased TER values (Fig. 5F–G), illustrating the favorable effects of empagliflozin on CMEC permeability and barrier function via the suppression of mitochondrial fission and subsequent oxidative stress. Moreover, empagliflozin also suppressed oxidative stress-mediated ICAM1 and VCAM1 upregulation while restored the contents of eNOS phosphorylation (Ser1177) (Fig. 5H–J). CMEC migration is vital for neovascularization. However, a trans- well assay revealed the impaired migration capacity of diabetic CMECs as evidenced by reduced ability of cells to translocate through mem- brane inserts in response to SDF-1 (Fig. 6A–B). Empagliflozin restored the chemotactic responses of CMECs by limiting fission because fission activation cancelled the positive actions of empagliflozin on CMEC migration. As F-actin is the key stress fiber that directly monitors CMEC mobilization, we speculated that blunted migration arose from fission- involved F-actin dysregulated homeostasis. Presence of fragmented mitochondria concurrently with disappearance of F-actin was sup- ported by the co-immunofluorescence of mitochondria and F-actin (Fig. 6C). Jasplakinolide (Jasp), a F-actin stabilizer was used to stabilize the actin cytoskeleton. The abolition of fission using mdivi1 preserved the filamentary structure of F-actin. Furthermore, the decrease in F-actin occurred in parallel with the accumulation of G-actin (Fig. 6D–F), an end-product of F-actin depo- lymerization, implying fission stimulated the disorder of F-actin synthesis and dissociation. The suppression of mitochondrial fission by empagliflozin reduced F-actin dissolution into G-actin, contributing to the recovery of CMEC migration capacity as evidenced by wound healing assay shown in Fig. 6G–H. Furthermore, these alterations in- duced by empagliflozin promoted neovascularization in vitro (Fig. 6I–J). If fission activation occurred via FCCP treatment, the con- structive effects of empagliflozin on angiogenesis were ablated (Fig. 6I–J). Together, these data confirmed the augmentation of CMEC migration and neovascularization by empagliflozin via interference with fission-associated F-actin collapse. To explore the possible mechanism by which empagliflozin coun- teracts excessive fission, Drp1 post-transcriptional modification was evaluated. Our results indicated that diabetes promoted Ser616 phos- phorylation on Drp1 although it reduced Ser637 phosphorylation (Fig. 7A–B), accompanied with a greater Drp1 accumulation on mi- tochondria. Empagliflozin promoted Ser637 but attenuated Ser616 phosphorylation of Drp1 in diabetes, leading to a lower mitochondrial Drp1 content (Fig. 7A–C). Next, given the key role of AMPK in the regulation of Drp1 phosphorylation, levels of AMP, ATP and AMPK activation were monitored. First, our data revealed suppressed AMPK phosphorylation in diabetes, the effect of which was reversed by em- pagliflozin (Fig. 7A–D). Moreover, empagliflozin also elevated the AMP/ATP ratio, favoring activation of AMPK. These data suggested that empagliflozin may promote AMPK activation via regulation of cellular AMP/ATP contents (Fig. 7E). The AMPK pathways activator and in- hibitor, AICAR (AI) and compound C (cC), respectively, were used as the positive and negative controls. AICAR promoted AMPK phosphor- ylation in diabetic group, which was accompanied with increased phosphorylation of Drp1S637 and reduced phosphorylation of Drp1S616 (Fig. 7A–D). In contrast, AMPK inhibition by compound C ameliorated the effects of empagliflozin on Drp1 regulation (Fig. 7A–D). These data suggested that empagliflozin balances Drp1 phosphorylation at both Ser616 and Ser637 residues through AMPK activation, eventually re- sulting in the failure of Drp1 recruitment to the mitochondria. 4.Discussion Pharmacological intervention targeting SGLT has recently emerged as a novel therapeutic option for diabetes [7,35]. Empagliflozin, a specific SGLT2 inhibitor, represents a promising new class of glucose- lowering drugs to speed up glucose excretion in urine independent of insulin. In addition to its well-established glycemic effect, ample evi- dence has indicated the cardioprotective potential of empagliflozin. The salient findings from our present study revealed that empagliflozin is capable of improving cardiac microvascular perfusion, barrier function, microvessel density, eNOS phosphorylation, endothelial-dependent re- laxation and CMEC survival. Our study indicated that empagliflozin may exert its beneficial effects through inhibition of diabetes-induced mitochondrial fission in an AMPK-dependent manner. With AMPK ac- tivation, empagliflozin effectively inhibited Drp1 activation and sub- sequently mitochondrial fission. On one hand, empagliflozin-induced decrease in fission may delay CMEC senescence by suppressing mtROS oxidative stress, leading to improved CMEC viability and barrier func- tion. On the other hand, empagliflozin-induced endothelial migration as a result of F-actin homeostasis may promote angiogenesis (Fig. 8). During diabetes progression, endothelial damage is detectable early on in the course of disease progression compared with the pathological changes occurred in cardiomyocytes [36], even before overt hy- perglycemia ensues. In the present study, we provided evidence for the first time supporting a direct protective role for empagliflozin in dia- betic microvascular injury through two distinct mechanisms namely alleviation of endothelial dysfunction and vascular remodeling. First, empagliflozin improved eNOS phosphorylation and barrier function, which alleviated luminal stenosis and inflammatory cell or erythrocyte attachment on microvessels. Second, empagliflozin increased CMEC survival and delayed vascular fibrosis, which greatly alleviated vascular degeneration and enriched microvessel density. Through these me- chanisms, empagliflozin increased blood circulation and cardiac per- fusion. Unlike great vessels possessing extensive nerve distribution, cardiac microvascular vessels modify their contraction and relaxation by balancing metabolic substances and eNOS phosphorylation [37]. The regulatory role of empagliflozin on eNOS phosphorylation is con- solidated by its action on vasomotor tone. Considerable evidence has documented the ability of empagliflozin to reduce SBP through facil- itating osmotic diuresis [38]. Our study further bolstered the role of empagliflozin in microvascular diastolic response by promoting eNOS phosphorylation. In addition, beneficial effects of empagliflozin on vascular remodeling was noted, characterized by improved CMEC sur- vival, lower levels of inflammatory related proteins, a thinner basement membrane, and decreased collagen fibrosis. These findings are re- miniscent of cardiovascular benefits of empagliflozin against myo- cardial remodeling and heart failure [39]. Taken together, our data add to previous evidence supporting the defensive and regulatory actions of empagliflozin on cardiac structure and components. In our study, we confirmed the regulatory role of mitochondrial fission in endothelial cell senescence and migration via mtROS over- production and F-actin degradation, respectively. This is supported by a number of experimental evidence. First, superoxide overproduction may serve as a unifying mechanism for diabetic microvascular com- plications [40]. Our previous study identified Nox4 as a source for in- tracellular ROS overproduction [41]. In the present work, fission was associated with an intracellular ROS outbreak via the induction of mtROS. The mechanism underlying this process involved fission-asso- ciated mPTP opening and membrane potential collapse, leading to ex- cessive electron leakage [42]. However, whether enhanced mitochon- drial fission triggers other ROS sources, such as Nox1, Nox2, and Nox4, deserves further scrutiny. Second, our findings suggested that fission led to F-actin depolymerization into G-actin, contributing to the in- ability of CMECs to migrate in response to SDF-1, representing an ul- timate obstacle in the formation of new micro-vessels. Indeed, succes- sive mitochondrial fission was dependent on the cascade of event among Drp1, Drp1 receptor and stress fibers [43]. It was reported that transient accumulation of intracellular F-actin on the mitochondrial surface is a prerequisite for mitochondrial division [44]. Under phy- siological conditions, F-actin is regularly distributed in certain parts of the cytoplasm that control microvascular deformation and vascular- ization [34,45]. When mitochondrial fission is initiated, F-actin first decomposes into G-actin that is then reassembled into F-actin at the outer mitochondrial membrane promote the formation of a contractile ring with the help of Drp1 and its receptor [46]. Considering the in- dispensable nature of F-actin in fission [47,48], we argue that excessive fission would consume large amounts of cytoplasmic F-actin and also cause the uneven distribution of F-actin [46], ultimately resulting in the dysregulated F-actin homeostasis and impaired migration. More evi- dence is needed to support this speculation. The suppression of fission by empagliflozin liberates oxidative stress and supports F-actin home- ostasis, which hinders cellular age and reverses vascularization po- tentiality. Here we provided the first piece of evidence indicating empagliflozin inhibited fission via an AMPK/Drp1-dependent mechanism. Notably, considerable disagreements exist with regards to the effect of AMPK signaling since both inhibitory and stimulatory roles of AMPK have been reported for mitochondrial fission. Previous studies have demonstrated that AMPK, a sensor of cellular energy shortage, pro- motes mitochondrial fission by activation of Mff, the receptor for Drp1 [49,50]. Nonetheless, other reports have argued that activation of AMPK represses mitochondrial fission through downregulation of Drp1 [51,52]. In aortic endothelial cells, Drp1 and mitochondrial fission are inactivated by AMPK-exerted phosphorylation at Ser637 [20]. More- over, our previous studies reconfirmed that AMPK activation was as- sociated with fission inhibition via suppression of Drp1 [21]. Given the complexity of mitochondrial fission, and existence of several factors in fission regulation apart from Drp1 [53], the precise role of AMPK in the control of fission activation deserves further investigation. Moreover, other signaling pathways may possibly be involved in empagliflozin- related mitochondrial homeostasis. Our previous finding suggested that oxidative stress-induced endothelial mitochondrial damage is resulted from IP3R-triggered cellular overload and subsequently mitochondrial calcium elevation via MCU (mitochondrial calcium uniporter) [54]. Whether IP3R-related calcium homeostasis is regulated by empagli- flozin is unknown. Besides, hyperglycemia-triggered mitochondrial fission may be lessened by mitophagy, a repair system for the mi- tochondrial homeostasis via timely clearance of the mitochondrial debris [15,53]. Although careful scrutiny has identified mitophagy as an endogenous defender of mitochondrial damage in diabetes [55,56], whether empagliflozin is capable of using the mitophagy machinery to disrupt the mitochondrial fission under diabetes remains unknown. Further study is warranted to verify such concept. The clinical implication that can be drawn from our study is multifold. First, our data provided compelling evidence for the clinical application of empagliflozin in diabetic patients with cardiac micro- vascular dysfunction. Although ample clinical studies have confirmed that empagliflozin can reduce the risk of adverse cardiovascular events in patients with diabetes [57], little information is available with regards to the protective mechanisms involved [58]. In this study, we observed the cardio-protective role of empagliflozin beyond its hypoglycemic effect. Second, empagliflozin regulates the mitochondrial function via an-AMPK-dependent manner based on our experimental findings, offering survival advantage for the cardiac microvascular endothelial cells. Through the improved cardiac blood flow supply, empagliflozin retards the vascular complications and sustains cardiac function in the context of diabetes. To this end, empagliflozin can be considered as a cardiac microvascular-protection drug to maintain cardiac circulatory TPH104m function and structure upon hyperglycemic insult. In conclusion, empagliflozin alleviated diabetic cardiac microvascular injury by inhibiting mitochondrial fission via the activation of AMPK signaling pathways. Further study is warranted to explore the clinical value of empagliflozin in diabetic vasculopathy.