AMPK signaling pathway

Journal Pre-proof Empagliflozin attenuates ischemia and reperfusion injury through LKB1/AMPK signaling pathway Qingguo Lu, Jia Liu, Xuan Li, Xiaodong Sun, Jingwen Zhang, Di Ren, Nanwei Tong, Ji Li PII:

S0303-7207(19)30344-2

DOI:

https://doi.org/10.1016/j.mce.2019.110642

Reference:

MCE 110642

To appear in:

Molecular and Cellular Endocrinology

Received Date: 27 September 2019 Revised Date:

6 November 2019

Accepted Date: 7 November 2019

Please cite this article as: Lu, Q., Liu, J., Li, X., Sun, X., Zhang, J., Ren, D., Tong, N., Li, J., Empagliflozin attenuates ischemia and reperfusion injury through LKB1/AMPK signaling pathway, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.110642. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Empagliflozin Attenuates Ischemia and Reperfusion Injury Through LKB1/AMPK Signaling Pathway

Qingguo Lua,b, Jia Liub,c, Xuan Lib, Xiaodong Sunb, Jingwen Zhangb,c, Di Renb,c, Nanwei Tonga, Ji Lib,c,* a

Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, 610041; bDepartment of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS 39216; cDepartment of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612

Short Title: Empagliflozin and Cardiac Ischemic Insult

*

Corresponding to: Ji Li, Ph.D., Department of Surgery, Morsani College of Medicine,

University of South Florida, 12901 Bruce B. Downs Blvd, MDC 110, Tampa, FL 33612. Tel: (813) 974-4917, Email: [email protected]

1

Abstract

The beneficial effects of empagliflozin (EMPA) on cardiac functions during ischemia and reperfusion were characterized. The contractile functions of isolated cardiomyocytes from adult C57BL/6J mice were determined with IonOptix SoftEdgeMyoCam system. The mitochondrial superoxide production was measured by MitoSOX fluorescent probe. The ex vivo isolated heart perfusion system was used to determine the pharmacological effects of EMPA on heart’s contractile functions under both physiological and pathological conditions. The in vivo regional myocardial ischemia and reperfusion by ligation of left artery coronary artery descending (LAD) was used to measure the myocardial infarction caused by ischemia and reperfusion with or without EMPA treatment. The results demonstrated that EMPA treatment significantly improves cardiomyocyte contractility under hypoxia conditions and augments the post-ischemic recovery in the ex vivo heart perfusion system. Furthermore, the in vivo myocardial infarction measurement shows that EMPA treatment significantly reduce myocardial infarct size caused by ischemia and reperfusion. The biochemical analysis demonstrated that EMPA can trigger cardiac AMPK signaling pathway and attenuate mitochondrial superoxide production under hypoxia and reoxygenation conditions. In conclusion, EMPA can trigger AMPK signaling pathways and modulate myocardial contractility and reduce myocardial infarct size caused by ischemia and reperfusion independent of hypoglycemic effect. The results for the first time demonstrate that the activation of AMPK by EMPA could one reason about EMPA’s beneficial effects on heart disease.

Key words: Empagliflozin, Ischemia (hypoxia)/reperfusion injury, Non-diabetic mice, AMPK

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1.

Introduction

Diabetes is considered as a significant risk factor for cardiovascular disease (CVD), the leading cause of global mortality(Mazzone, 2010,Rawshani, Sattar, Franzen et al., 2018). Aims of diabetes treatment are multiple risk factors control, such as hyperglycemia, hyperlipidemia and hypertension, etc. At present, many kinds of hypoglycemic drugs are available for clinical use. As a request by the US Food and Drug Administration (FDA), clinical research evidence of cardiovascular (CV) safety must be submitted by the pharmaceutical industry before the approval of a novel antidiabetic agent (FDA, 2008). Sodium-glucose cotransporter 2 (SGLT2) inhibitors are new class of antidiabetic agents which act by selectively inhibiting SGLT2 of the renal proximal tubule, with a consequent of increase in urinary excretion of glucose, reducing blood glucose concentration independent of insulin. Canagliflozin, dapagliflozin, empagliflozin (EMPA) and ertugliflozin of this family have been approved by FDA. Encouraging evidence of CV protection effects from several large clinical studies of SGLT2 inhibitors have been published(Zinman, Wanner, Lachin et al., 2015,Kosiborod, Cavender, Fu et al., 2017,Neal, Perkovic, Mahaffey et al., 2017,Wiviott, Raz, Bonaca et al., 2018). EMPA REG OUTCOME (Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes)is the first study for CV protection, which suggested that EMPA exerts a 38% risk reduction in death from CV causes, 32% risk reduction death from any cause, and 35% reduction on risk of hospitalization for HF(Zinman et al., 2015,Kosiborod et al., 2017,Neal et al., 2017,Wiviott et al., 2018). It is noteworthy that this effect of EMPA is independent of its hypoglycemic effect. A new indication has been approved by FDA for EMPA to reduce the risk of CVD death in adult patients with T2DM and CVD in 2016 (FDA, 2016). At present, many studies are trying to explore the mechanism of CV protection of SGLT2 inhibitors independent of hypoglycemia effect. Most of the research are focusing on energy metabolism, inflammation, oxidative stress, myocardial fibrosis and electrolyte homeostasis (Bertero, Prates Roma, Ameri et al., 2018). Multiple studies had been reported that EMPA could improve myocardial fibrosis in obese and 3

diabetic mice (Lee, Chang and Lin, 2017,Ye, Bajaj, Yang et al., 2017) and also play the role of anti-oxidative stress and anti-apoptosis (Lin, Koibuchi, Hasegawa et al., 2014,Zhou and Wu, 2017). Meanwhile, EMPA was found to decrease the infarct area under ischemia/reperfusion (I/R) stress (Andreadou, Efentakis, Balafas et al., 2017) and improve diastolic function of the left ventricle (LV) in diabetic mice (Hammoudi, Jeong, Singh et al., 2017). Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an important serine and threonine protein kinase, which plays an important role in cell energy balance(Hardie, 2004). Increasing the ratio between intracellular adenosine monophosphate (AMP) and adenosine triphosphate (ATP) will activate AMPK by phosphorylation(Hardie and Carling, 1997). In addition, liver kinase B1 (LKB1), calmodulin-dependent protein kinase kinase β (CaMKKβ), and AMPK kinase (AMPKK) are all upstream activators. AMPK also plays an important role in reducing oxidative stress, regulating autophagy, and anti-apoptosis of cardiomyocytes(Bertrand, Ginion, Beauloye et al., 2006,He, Zhu, Li et al., 2013). It has been reported that EMPA can protect the heart by activating AMPK in cardiac microvascular endothelial cells (CMEC) of diabetic mice(Zhou, Wang, Zhu et al., 2018). Canagliflozin can regulate energy metabolism via AMPK activation by inhibiting Complex I in the respiratory chain in HEK-293 cells and hepatocytes(Hawley, Ford, Smith et al., 2016). The basic research on SGLT2 inhibitors was mostly focused on animal models of diabetes and/or obesity. To date, it is unclear if SGLT2 inhibitors could offer protection against hypoxia/reoxygenation (H/R) or I/R injury in non-diabetic and non-obese model through AMPK signaling pathway. This study aims to explore whether short-term treatment of EMPA could protect against H/R or I/R injury in healthy young heart. In this study, we selected healthy young male mice as research objects, explored whether short-term administration of EMPA displays protective effect on cardiomyocytes and cardiac function, and the influence of myocardial infarction (MI) area in vitro and in vivo under H/R and I/R stress conditions. At the same time, we tried to further explore whether AMPK related signaling pathway was involved in this mechanism. 4

2. Materials and Methods Young (12weeks) male C57BL/6J mice were obtained from Jackson Laboratory. EMPA and compound C were purchased from Sigma-Aldrich and ApexBio respectively. All animal activity protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center in this study.

2.1. Cardiomyocytes isolation from mice Heparin IV (Fresenius Kabi) for anticoagulation (Ma and Li, 2015,Li, Liu, Hu et al., 2018)was given by intraperitoneal injection with 1000 units/kg 10 min before the experiment. Mice were killed after anaesthesia with 2%–3% isoflurane and 100% O2. The hearts of mice were excised, then cannulated by aorta and connected to the cardiomyocyte perfusion apparatus (Radnoti). The heart was perfused at 37℃ with a Ca2+ free based buffer (pH 7.2) containing: 135 mM NaCl, 4 mM KCl, 1mM MgCl2, 10 mM HEPES, 0.33 mM NaH2PO4, 10 mM glucose, 10mM 2, 3-butanedione monoxime (Sigma), and 5 mM taurine(Sigma) that was bubbled with 95% O2 and 5% CO2.The heart was digested in 25 ml perfusion buffer with 0.3 mg/g body weight collagenase D (Roche), 0.4 mg/g body weight collagenase B (Roche), and 0.05 mg/g body weight protease type XIV (Sigma) dissolved after 3–5 min of recycling. After digested completely, the heart was removed, torn with tweezers and blown gently, then filtered to get the isolated cardiomyocytes.

2.2. Time gradient experiment of cardiomyocytes under normoxia condition Isolated cardiomyocytes were divided into two groups with vehicle (PBS:DMSO=105:1) and EMPA of 0.5µM respectively. Each group was further divided into five subgroups, which were cultured at a time gradient of 10 min for 50 min under normoxia condition. Samples of cardiomyocytes were harvested at every time points for detecting the level of related signal pathway molecular.

Treatment of cardiomyocytes with hypoxia and reoxygenation 5

Isolated cardiomyocytes were treated with vehicle or 0.5µM EMPA and placed in a nitrogen chamber containing 95%N2 and 5%CO2 for 20min to induce hypoxia. Then they were transfered from the nitrogen chamber to normoxia incubator and allowed to reoxygenate for 20 min. The control groups of cardiomyocytes under normoxia condition were treated with vehicle or 0.5µM EMPA and placed in normal incubator for the same time.

2.3. Groups and drug administration of cardiomyocytes in H/R experiment Isolated cardiomyocytes of mice were randomly divided into six groups according to treatment and conditions (normoxia or H/R): baseline with vehicle (B+V), baseline with EMPA (B+E), H/R with vehicle (H/R+V), H/R with EMPA (H/R+E), H/R with Compound C (H/R+C), H/R with Compound C and EMPA (H/R+C+E). The concentration of EMPA and Compound C were 0.5µM and 20µM respectively according to pharmacokinetics and references (Andreadou et al., 2017,Panchapakesan, Pegg, Gross et al., 2013,Tahara, Takasu, Yokono et al., 2016).

2.4. Immunoblotting The way of immunoblotting was previously described in our publications (Li et al., 2018,Quan, Sun, Wang et al., 2017). Briefly, total protein was extracted by lysis buffer from the cardiomyocytes samples of time gradient and H/R experiments. Concentration of protein was determined by Bradford dye binding method (Bio-Rad). Target proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Millipore). Primary rabbit antibodies LKB1 (3047s), phosphorylated LKB1 (p-LKB1)(3482s), AMPKα (2532s), phosphorylated AMPKα (p-AMPKα)(2535s), acetyl coenzyme A carboxylase (ACC)(3661s), phosphorylated ACC (p-ACC)(3676s), peroxisome proliferator-activated receptor co-stimulator 1α (PGC1α)(3820s) and horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody were purchased from Cell Signaling Technology.

2.5. Contractility measurement of mice cardiomyocytes 6

Extracellular Ca2+ solution was used back to the isolated cardiomyocytes gradually from 0.06 mM to a final concentration of 1.2 mM with the interval incubation time of 15 min for each Ca2+concentration. Then the cells were washed by contraction buffer (10mM glucose,1 mM CaCl2, HEPES buffer to 25ml) for three times. The contractility of cardiomyocytes was measured by SoftEdgeMyocam system (IonOptix Corporation). Cardiomyocytes were placed in a chamber and stimulated with a supra-threshold voltage at a frequency of 1 Hz (Gruntzig, 1978,Moshal, Kumar, Tyagi et al., 2009). The data of changes in sarcomere length, duration of shortening and re-lengthening were recorded and analyzed by IonOptix SoftEdge software. The following parameters can be used to evaluate the cardiomyocytes: resting sarcomere length, the maximum velocity of shortening, maximum contraction amplitude.

2.6. Mitochondrial superoxide measurements of cardiomyocytes Mitochondrial superoxide production, a type of reactive oxygen species (ROS) of cardiomyocytes, was measured by MitoSOX Red (Invitrogen). The cells were loaded with MitoSOX Red (5 µM) for 10 min at 37℃ protected from light, and then washed. Fluorescence microscope (excitation at 514 nm and measuring the emitted light at 585 nm) was employed to obtain the images of cardiomyocytes.

2.7. Isolated heart perfusion (Langendorff) Methods of isolated heart perfusion were described before in our publications(Li et al., 2018,Quan et al., 2017). Briefly, mice were anesthetized with isoflurane (2–3%) and the hearts were excised. After aortic retrograde intubation, isolated hearts were perfused in the Langendorff system at 37°C with Kre bs-Henseleit (K-H) buffer(Morrison, Chen, Wang et al., 2015,Yang, Sun, Quan et al., 2016), which containing: 118 mM NaCl,4.75mM KCl, 0.94mM KH2PO4, 1.2mM MgSO4, 25mM NaHCO3, 1.4mM CaCl2, 7 mM glucose, 0.4 mM oleate, 1% bovine serum albumin (BSA),10 mU/ml insulin. Hearts were perfused for 20 min at a flow speed of 4ml/min, followed by turning off the pump to simulate global ischemia for 40 min or 30 min and then reperfusion for 30 min. A balloon filled by fluid was inserted into the LV cavity and 7

connected to the Chart 8 system (AdInstruments, Colorado Springs) to measure and record heart rate and LV developed pressure, then we calculated the systolic blood pressure and rate-pressure product (RPP). The balloon was inflated to achieve a baseline LV end-diastolic pressure of 5 mmHg that was kept constant during I/R (Morrison et al., 2015,Wang, Tong, Yan et al., 2013).

2.8. Groups and drug administration of isolated heart perfusionexperiment Based on the object of this study, we divided the isolated hearts as four groups: I/R with vehicle (PBS: DMSO = 20000:1) (I/R+V), I/R with EMPA (I/R+E), I/R with Compound C (I/R+C), I/R with Compound C and EMPA (I/R+C+E). EMPA was prepared with K-H buffer at a final concentration of 2.5 µM and perfused into the heart. Compound C was given to the mice by intraperitoneal injection with doses of 1mg/kg body weight one hour before the experiment. Drug administration methods and doses are based on previous studies and pharmacokinetics(Tahara et al., 2016).

2.9. Infarct area measurement after isolated heart perfusion. After 30min of reperfusion, the hearts were removed from the Langendorff system and perfused with 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution by syringe and immersed the whole heart in TTC solution for 10 min. Then the hearts were left in 10% formalin for 48 h and cut into slices about 1 mm thickness and photographed with a Leica microscope (Leica Microsystems). TTC can stain normal myocardial tissue red, while the infarct area can't be stained and appear white. We analyzed infarct area with Image J software (National Institutes of Health)(Li et al., 2018,Morrison, Yan, Tong et al., 2011) and calculate the percentage of infarct size to the whole heart slice for comparation between groups.

2.10.

Ischemia/reperfusion treatment

Mice were anesthetized with 2%–3% isoflurane and placed on a heating pad to keep body warm. A ventilator (Harvard Apparatus) was connected after endotracheal intubation. Left lateral thoracotomy was performed to expose the heart under the 8

operating microscope. We occluded the left anterior descending artery (LAD) by 8–0 nylon suture with a polyethylene tube in the surgical knot to protect the LAD from injury by suture during ligation. ST-segment elevation of electrocardiogram (ECG) and regional blanching of the left ventricle were signs of successful operation. After regional ischemia for 45 min, we closed the chest and suture followed by 24h reperfusion.

2.11.

Groups and drug administration of mice in I/R experiment

The mice were given vehicle, EMPA or Compound C everyday three days before operation. Administration mode of EMPA was gavage with the dose of 10mg/kg body weight. Compound C was given by intraperitoneal injection with the dose of 1mg/kg body weight. Pharmacokinetics and previous literature were consulted for the administration and doses of all drugs (Andreadou et al., 2017,Zhou et al., 2018,Tahara et al., 2016,Cheng, Chen, Li et al., 2016). We divided all the mice as six groups: sham with vehicle (sham+V), sham with EMPA (sham+E), I/R with vehicle (I/R+V), I/R with EMPA (I/R+E), I/R with Compound C (I/R+C), I/R with Compound C and EMPA (I/R+C+E).

2.12.

Echocardiography

After I/R treatment, mice were anesthetized (isoflurane) and transthoracic B-mode echocardiography (Vevo 3100, Visualsonics) were performed to measure cardiac function. We obtain the data of averaged LV ejection fraction (LVEF) and fraction shortening (FS) of all mice by Simpson’s measurements.

2.13.

Infarct size assessment in vivo

Mice were anesthetized and the hearts were excised immediately after 24h of reperfusion. TTC and Evans blue dye were used to stain the hearts, and then they were kept in 10% formalin for 48h. We cut the stained heart into slices of 1mm thickness, and took photos with a Leica microscope (Leica Microsystems). Image J software (National Institutes of Health)(Li et al., 2018,Morrison et al., 2011)was also 9

employed for analysis. The region stained blue from Evans Blue suggested the non-ischemic region (safe area), and the red area stained by TTC represented the ischemic area at risk (AAR). The infarcted area (INF) appeared white because it could not be stained by TTC. Finally, we calculated the proportion of myocardial INF to AAR and carried on statistical analysis.

2.14.

Histological examination

We selected hearts randomly from each groups immediately after reperfusion in vivo. All hearts were excised and washed with PBS to remove residual blood and immersed in 10% formalin overnight, then embedded in paraffin. Hearts from each group were cut into slices of 5µm thickness serially and stained with hematoxylin and eosin stain (H&E) for routine histologic examination (Sun, Quan, Wang et al., 2016,Zhang, Zhao, Quan et al., 2017).

2.15.

Statistical analysis

Data were expressed as means ± standard error of the means (SEM). Two-tailed Student’s t test and one-way ANOVA with Tukey’s test for post hoc comparisons were used by Prism 7.0 (GraphPad Software). p< 0.05 was considered as significant difference.

3. Results

3.1. EMPA activates LKB1/AMPK signaling pathway in isolated mice cardiomyocytes under normoxia and H/R condition We got the samples of 11 subgroups (including 0 min time point) with vehicle or EMPA in time gradient experiment under normoxia state. Western-blot showed that phosphorylated activation of LKB1 and AMPK in EMPA group were more significant than that in vehicle group at corresponding same time point (p< 0.05) (Figs. 1A-C). Although LKB1 in the vehicle group was activated by phosphorylation at 10 and 20 10

min, statistical analysis showed that it was more activated at the same time point in the EMPA group (p< 0.05) (Figs. 1A and B). Phosphorylated LKB1 (p-LKB1) and AMPK (p-AMPK) reached their peak value at 20-30 min of time gradient (Figs. 1B and C). Phosphorylated-ACC (p-ACC) and PGC1α level downstream also increased (Figs. 1D and E) (p< 0.05). Therefore, EMPA can activate LKB1/AMPK and downstream pathways in baseline state. Under the condition of hypoxia for 20 min and reoxygenation for 20 min, we found that LKB1 and AMPK were activated in vehicle group and EMPA group (Figs. 2A-C), the activation of LKB1/AMPK was more significant and lasted longer after EMPA treatment under H/R stress conditions (p< 0.05) (Figs. 2B and C). In H/R+C+E group, AMPK phosphorylation was inhibited by Compound C, however, LKB1 was still significantly activated by EMPA. This further confirms that the activation of AMPK by EMPA is via the upstream molecular-LKB1 in cardiomyocytes of mice. In addition, we also found higher levels of p-ACC and PGC1α in EMPA groups than other groups (p< 0.05) (Figs. 2D and E). Therefore, AMPK can play the roles of protective effect on cardiomyocytes through ACC and PGC1α downstream pathway.

3.2. EMPA improves contractility of isolated cardiomyocytes of mice after H/R stress conditions SoftEdge Myocam system and Ion Wizard software were employed to record and analyze the data of contractile function of cardiomyocytes in H/R experiment (Fig. 3). The results showed that the length of sarcomeres at rest in each group was similar (p> 0.05) (Fig. 3A), indicating that the basal state of all cardiomyocytes was consistent in each group. Under baseline condition, the maximum contraction velocity and amplitude were slightly decreased after treatment of 0.5µM EMPA (p< 0.05). Under H/R stress, the maximal contraction velocity and amplitude of cardiomyocytes were significantly lower than those of baseline state (p< 0.01). But after adding 0.5 µM EMPA to the cardiomyocytes at the same time of H/R stress, the contractile function was improved (p<0.01), but still lower than that of baseline state. Compound C inhibited the activity of AMPK under H/R stress and further damaged the contractile 11

function of isolated cardiomyocytes, which could not be compensated by EMPA (p >0.05) (Figs. 3B and C). These data indicated that EMPA can improve the contractile function of mice cardiomyocytes under H/R stress condition, and AMPK related signal pathway is essential for this effect. We summarized the myocardial sarcomeres shortening of each group in Fig. 3D.

3.3. EMPA reduces mitochondrial superoxide production in isolated cardiomyocytes after H/R stress There were no differences in superoxide production between the vehicle and EMPA group (p> 0.05) under normoxia conditions. Superoxide production increased in H/R group, which can be attenuated by 0.5 µM EMPA (p < 0.05). But they increased significantly after adding Compound C to cardiomyocytes under H/R stress conditions, and EMPA had no significant effect on reducing superoxide in the presence of Compound C(p> 0.05) (Figs. 4A and B). The results indicated that EMPA could reduce the production of superoxide through AMPK pathway, thus exerting the role of anti-oxidant stress under H/R stress conditions.

3.4. EMPA improve the systolic function of heart after I/R in isolated heart perfusion system In the isolated heart perfusion system (Langendorff), vehicle, EMPA and Compound C were perfused to isolated hearts by K-H buffer, respectively. The vehicle and EMPA groups received global ischemia for 40min, then reperfusion for 30min. We found that RPP of EMPA group was significantly higher than that of vehicle group during I/R stress condition (p＜0.05) (Fig. 5A). We had tried to make the isolated hearts ischemia for 40 min in Compound C group as well, unfortunately, the hearts could not regain beating after reperfusion. Therefore, ischemia time of Compound C and Compound C+EMPA groups decreased to 30min, the reperfusion time is still 30min. It was found that there were no significant differences of RPP between the two groups (p> 0.05) under I/R conditions (Fig. 5C). These data show that EMPA could improve the systolic function of isolated heart under I/R condition in the Langendorff system, in 12

which AMPK signaling pathways were involved in this role.

3.5. EMPA reduce the area of myocardial infarction (MI) after I/R in Langendorff system of mice We calculated the percentage of the MI area to the total area of heart in each slice, and compared the proportion of the MI area in each group. The statistical results showed that the percentage of MI area in EMPA group were significantly less than that in vehicle group (p< 0.05) (Figs. 5B). There were no significant differences in the MI area between Compound C and EMPA+Compound C group (p> 0.05) (Figs. 5D). Although the ischemic time of the two groups was shorter, the MI area percentage of them was still larger than that of the vehicle and EMPA groups (p< 0.05). It is suggested that EMPA can reduce the area of MI after I/R in the Langendorff system. When Compound C was used to inhibit AMPK at the same time, EMPA could not play a role in reducing the area of MI, indicating that this role of EMPA was achieved at least partly through the activation of AMPK pathway.

3.6. EMPA improve systolic function of the heart after I/R in vivo Echocardiography was performed before and after LAD surgery (Fig.6A). No significant change in blood glucose was recorded before and after EMPA administration, which excluded the potential hypoglycemic effect of EMPA on cardiac function. LVEF and FS were used as indicators of cardiac systolic function. There was no significant difference in preoperative cardiac systolic function among all groups. LVEF and FS in Sham (Vehicle and EMPA) groups also did not change significantly before and after operation. LVEF and FS in I/R + vehicle group decreased significantly after I/R stress compared with preoperative state, while EMPA can improve LVEF, but not FS, compared with vehicle group under I/R stress (p< 0.05) (Figs.6B and 6C). The decrease of LVEF of mice in I/R + Compound C group was more severe than that in I/R + vehicle group(Fig.6B). EMPA also could not significantly ameliorate the LVEF of mice when they were treated with Compound C under I/R stress (Fig.6B). All the results from echocardiography indicated that EMPA can maintain the systolic function 13

of mice after I/R stress, and this protective effect is largely achieved through AMPK and downstream signal pathways. EMPA can reduce the area of MI after I/R stress in vivo After stained by TTC and Evans blue, we cut the heart into slices of 1mm thickness and took photos (Fig.7A). The proportion of AAR and MI area to the total area of cardiac slices was no statistical differnce in each group (p> 0.05) (Fig.7B), which indicated that the degree of ischemic stress was the same in each group. Statistical analysis results showed that the percentage of MI area in EMPA group was significantly less than that in other three groups (p<0.05) (Fig.7C). There was no difference between Compound C and EMPA+Compound C groups (p>0.05), and the MI area of these two groups was larger than that of vehicle and EMPA groups (p<0.05) (Fig.7C). These results suggested that EMPA can reduce the area of MI in mice after I/R stress in vivo, but it couldn't work when AMPK is inhibited, which indicated that the effect of EMPA is at least partly achieved by activation of AMPK. This is consistent with the results of isolated heart perfusion experiment.

3.7. Histological examination Histological sections and HE staining were performed on the heart tissues of each group (Fig.8). The results showed that there was no significant change in myocardial histology in sham + EMPA group compared with sham + vehicle group. In I/R + vehicle group, there was obvious infiltration of inflammatory cells (most of them are neutrophils) in the interstitial space of myocardial tissue near the epicardium. After EMPA treatment during I/R stress condition, the infiltration of inflammatory cells was alleviated. We could observe more severe infiltration of inflammatory cells in I/R + Compound C group than that in I/R+ vehicle group and EMPA could't improve this pathological change under this condition. In addition, no significant histological changes were observed in cardiomyocytes, then ucleus, cytoplasm and interstitial cells in each group. These results indicated that EMPA may act as an anti-acute myocardial inflammation agent through AMPK pathway under I/R stress. 4. Discussion 14

Ischemic heart disease is the leading cause of death worldwide, and diabetes is an important triggering factor. Therefore, the treatment of diabetes is not simply to control blood glucose, but to control multiple risk factors such as blood pressure, serum lipid, uric acid, etc. in order to reduce the morbidity of CVD, other complications and mortality ultimately. At present, the safety requirements for hypoglycemic drugs is also increasing, at least not to increase the risk of CVD, it would be better if there is CV benefits at the same time. Among the hypoglycemic drugs on market, metformin and glucagon-likepeptide1 (GLP-1) agonists have been proved to have CV protective effects. With the increasing request of FDA on CV risk, more hypoglycemic drugs with CV benefits will appear. SGLT2 inhibitors have been shown to be beneficial in heart failure, ischemic heart disease and all-cause mortality in several large clinical trials(Zinman et al., 2015,Kosiborod et al., 2017,Wiviott et al., 2018). After clinical analysis, the protective effect of SGLT2 inhibitors on heart may be related to lowering blood pressure, weight loss, decreasing serum uric acid level, osmotic diuresis, reducing volume load and hemodynamic changes, etc. Further molecular mechanism is still in the exploratory stage. AMPK is an important energy regulator and protective factor in the heart. Our group and others have shown that AMPK is cardioprotective during ischemia by enhancing glucose uptake and glucose transporter 4 (GLUT4) translocation (Miller, Li, Leng et al., 2008), decreasing apoptosis, improving post-ischemic recovery, and limiting MI(Ma, Wang, Thomas et al., 2010,Russell, Li, Coven et al., 2004). Furthermore, our studies have also demonstrated that pharmacological activation of AMPK by activated protein C could protect the heart against I/R injury(Wang, Yang, Rezaie et al., 2011), and inhibit inflammatory responses during H/R by modulating a JNK-mediated NF-κB pathway(Chen, Li, Zhang et al., 2018). Few studies are focusing on the relation of SGLT2 inhibitors and AMPK now. As mentioned earlier, canagliflozin can activate AMPK in HEK-293 cells and hepatocytes (Hawley et al., 2016). It can also inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK dependent and independent mechanisms (Mancini, Boyd, Katwan et al., 2018). Dapagliflozin could attenuate myocardial inflammation, fibrosis, apoptosis, and 15

diabetic remodeling likely mediated through AMPK activation (Ye et al., 2017). EMPA alleviated diabetic cardiac microvascular endothelial cell (CMEC) injury by inhibiting mitochondrial fission via the activation of AMPK-Drp1 (Dynamin-related protein 1) signaling pathways, preserved cardiac CMEC barrier function through suppressed mitochondrial ROS production and subsequently oxidative stress to impede CMEC senescence (Zhou et al., 2018). So EMPA can be considered as a cardiac microvascular-protection drug to maintain cardiac circulatory function and structure upon hyperglycemic insult. In our study, we found that EMPA can improve the contractile function of isolated cardiomyocytes under H/R conditions and reduce the production of mitochondrial superoxide in vitro. In the Langendorff system, EMPA could increase RPP of isolated hearts and significantly reduce the MI area under I/R stress. Pretreatment with EMPA could significantly improve the LVEF of the mice heart under I/R conditions, and also attenuate the area of MI under this condition in vivo. In this study, there was no significant statistical difference in FS among each group, but the change trend was similar to LVEF, which may be related to the relatively small sample size of this study. Because LVEF is more stereoscopic and accurate by volume measurement, we prefer to use LVEF to represent the systolic function of the heart in this study. The effect of EMPA on reducing infarct size after I/R is very significant and intuitive, which is a very encouraging result. The improvement of cardiac function by EMPA in vitro and in vivo is also closely related to the reduction of MI area. EMPA may also has an anti-acute inflammatory effect under I/R conditions according to pathological section. Therefore, our findings are consistent with the results of the cardioprotective effects of EMPA in clinical studies. Myocardial H/R or I/R injury has been the focus of basic and clinical research. Main mechanisms are associated with increased production of free radicals[including ROS and Reactive Nitrogen Species (RNS)], calcium overload, inflammation and microcirculation disorders. The energy metabolism disorder caused by ischemia and hypoxia leads to the decrease of mitochondrial ATP synthesis, which is an important reason for the above pathophysiological processes. AMPK has been shown to protect 16

myocardial I/R or H/R injury by many studies(Ma et al., 2010,Russell et al., 2004,Wang et al., 2011). Based on our hypothesis, we tried to validate whether AMPK is involved in the cardioprotective effect of EMPA in our study. Western blot was used to detect the expression of the target protein in isolated cardiomyocytes, which avoided the influence of protein expression in other cardiac cells such as vascular endothelial cells and fibroblasts. The results showed that AMPK could be phosphorylated and activated in baseline, H/R and I/R state after EMPA treatment of 0.5 µm. Although ischemia or hypoxia can also activate AMPK, it may not completely compensate for the damage caused by H/R or I/R. AMPK is activated to a higher degree and lasts longer by adding exogenous activator EMPA, thus exerting its cardioprotective effect to a greater extent. To further verify the role of AMPK in cardioprotective effect of EMPA, we used the AMPK inhibitor Compound C. It was found that in the presence of Compound C, EMPA could not play its role in improving cardiomyocytes and cardiac contractile function, reducing superoxide production, attenuating infarct size and anti-acute inflammatory. These results further confirm that the protective effect of EMPA on heart may be achieved through AMPK signaling pathway. As we know, the increase of intracellular AMP/ATP ratio is an important cause of AMPK phosphorylation activation, and LKB1, CaMKKβ and AMPKK are the upstream activating factors of AMPK. In our study, we detected the upstream molecules of AMPK, and found that phosphorylated activation of LKB1 was consistent with that of AMPK, and LKB1 was still activated significantly when Compound C inhibited the activation of AMPK, which indicated that EMPA might activate AMPK through the upstream LKB1. So how does EMPA activate LKB1？Xie et al.(Xie, Dong, Zhang et al., 2006) found that the phosphorylation of LKB1 at Ser428 can enhance the ability of LKB1 to bind and activate AMPK. Protein kinase A (PKA) is the upstream molecule of LKB1, which can phosphorylate LKB1(Huang, Zhu, Chen et al., 2019). Our previous study found that Sirtuin 1 (SIRT1) can increase the deacetylation of LKB1 and increase its phosphorylation(Wang, Quan, Sun et al., 2018). Whether EMPA can directly activate LKB1 by phosphorylation or through PKA, SIRT1 etc. has 17

not been reported. We will carry out further research to reveal this mechanism in the future. At the same time, we detected some target molecules downstream of AMPK by Western blot, the results showed that PGC-1α and phosphorylated ACC levels in EMPA group increased significantly, which was consistent with AMPK activation. We know that AMPK/ACC signaling pathway is an important way to increase fatty acid oxidation and ATP production to improve energy metabolism. AMPK/PGC-1α pathway plays an important role in anti-oxidative stress injury. Energy metabolism disorder and oxidative stress are just important pathophysiological processes of H/R and I/R injury. The reduction of MI area by EMPA can also be explained by AMPK-related signaling pathways to improve myocardial energy metabolism and antioxidant stress, thereby increasing cell survival during I/R stress. Therefore, these results above suggest that the activation of LKB1/AMPK/ACC and LKB1/AMPK/PGC-1a signaling pathways is an important molecular mechanism for the cardioprotective effect of EMPA under H/R and I/R stress. An interesting result was found in cardiomyocyte experiments in vitro. At normoxia condition of baseline, the contractile function of cardiomyocytes decreased after adding 0.5 µM of EMPA compared with that of vehicle group, that is to say, negative inotropic effect was observed. Under H/R condition, the contractile function of cardiomyocytes after treatment of the same concentration of EMPA was stronger than that of H/R with vehicle group but still weaker than baseline. What is the reason? Baartscheer et al(Baartscheer, Schumacher, Wust et al., 2017) found that EMPA could inhibit Na+/H+ exchanger (NHE) in rat and rabbit cardiomyocytes at baseline state, reduce Na+ and Ca2+ in cytoplasm, increase Ca2+ in mitochondria, and show negative inotropic effect. This research can explain the negative inotropic effect of EMPA under baseline condition in our study. Therefore, we speculate that EMPA has a negative inotropic effect on cardiomyocytes mainly by inhibiting NHE at baseline. It can also be interpreted that EMPA may have an "energy-saving" effect under normoxia conditions. Once in H/R and I/R conditions, EMPA mainly increases ATP production through AMPK/ACC pathway, and anti-oxidative stress through AMPK/PGC-1a pathway to achieve myocardial protection and enhancement of 18

contractile function. Therefore, the role of EMPA in balancing energy metabolism under different conditions is vital for maintaining the function of cardiomyocytes. In addition, the most interesting feature of our study is the use of "non-diabetic normal mice model". The results from related clinical trials also indicate that the cardioprotective effect of SGLT2 inhibitors is probably independent of its hypoglycemic effect. Because the clinical indications of SGLT2 inhibitors are type 2 diabetes mellitus, the intervention population of clinical studies are all diabetic patients, and the animal models used in basic research are mostly diabetes or obesity. In our study, isolated cardiomyocyte and heart perfusion experiments in vitro excluded the hypoglycemic effect of EMPA completely, because the hypoglycemic target of EMPA is in the kidney. EMPA administration term is relatively short in vivo, and there is no significant change in blood glucose before and after treatment, which also excludes the effect of hypoglycemic effect. Therefore, the results of this study are important for evaluating the cardioprotective effects and mechanisms of EMPA independent of hypoglycemia. EMPA has been approved for a new indications for reducing CV mortality in type 2 diabetes with CVD (FDA, 2016).. The findings of this study may provide basic research evidence for further expanding the clinical indications of these drugs, and may play a major role in reducing some adverse outcomes of I/R injury in these patients after coronary angioplasty or reconstruction (e.g., coronary artery bypass grafting and percutaneous transluminal coronary angioplasty). At the same time, other studies have reported that EMPA may play a cardioprotective role through other signaling pathways. Zhou et al(Zhou and Wu, 2017) found that EMPA can protect the heart of diabetic rats by inhibiting apoptosis of cardiomyocytes induced by endoplasmic reticulum stress. A study of long-term EMPA treatment to obese mice indicated that EMPA improved myocardial function and reduced infarct size as well as improves redox regulation by decreasing inducible nitric oxide synthase (iNOS) expression and subsequently lipid peroxidation as shown by its surrogate marker malondialdehyde (MDA). The mechanisms of action implicated in the activation of signal transducer and activator of transcription 3 (STAT3) 19

pathway anti-oxidant and anti-inflammatory properties(Andreadou et al., 2017). Some studies suggested that EMPA could not activate AMPK (Andreadou et al., 2017,Hawley et al., 2016). We speculate that the different results may be related to the intervening time or concentration of EMPA and the animal model used. This is a preliminary study on the protective effect and mechanism of EMPA on cardiac H/R and I/R injury in non-diabetic normal mice with some limitations. Firstly, although we found that EMPA could activate LKB1/AMPK signaling pathway in cardiomyocytes of mice, the mechanism by which EMPA activates LKB1 requires further investigation. Secondly, the sample size is relatively small of isolated heart perfusion and in vivo experiments, which needs to be further increased. Thirdly, we only observed the possible anti-acute inflammatory effect of EMPA on pathological slices. Further experiments are needed to determine whether EMPA has this effect and its mechanism. In conclusion, EMPA can improve myocardial contractility and attenuate myocardial infarct size after H/R and I/R stress condition independent of hypoglycemic effect. The cardioprotective effect of EMAP may be achieved by LKB1/AMPK/ACC and LKB1/AMPK/PGC1α signaling pathways.

FUNDING The present study was supported by Sichuan Science and Technology Program [grant number 2019YJ0062], American Diabetes Association [grant number 1-17-IBS-296].

Conflict of Interest The authors declare that they have no conflict of interest.

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2784-2794. He, C., Zhu, H., Li, H., Zou, M. H., Xie, Z., 2013. Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes. Diabetes 62, 1270-1281. Huang, Y., Zhu, X., Chen, K., Lang, H., Zhang, Y., Hou, P., Ran, L., Zhou, M., Zheng, J., Yi, L., Mi, M., Zhang, Q., 2019. Resveratrol prevents sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress via the PKA/LKB1/AMPK pathway. Aging 11, 2217-2240. Kosiborod, M., Cavender, M. A., Fu, A. Z., Wilding, J. P., Khunti, K., Holl, R. W., Norhammar, A., Birkeland, K. I., Jorgensen, M. E., Thuresson, M., Arya, N., Bodegard, J., Hammar, N., Fenici, P., Investigators, C.-R., Study, G., 2017. Lower Risk of Heart Failure and Death in Patients Initiated on Sodium-Glucose Cotransporter-2 Inhibitors Versus Other Glucose-Lowering Drugs: The CVD-REAL Study (Comparative Effectiveness of Cardiovascular Outcomes in New Users of Sodium-Glucose Cotransporter-2 Inhibitors). Circulation 136, 249-259. Lee, T. M., Chang, N. C., Lin, S. Z., 2017. Dapagliflozin, a selective SGLT2 Inhibitor, attenuated cardiac fibrosis by regulating the macrophage polarization via STAT3 signaling in infarcted rat hearts. Free Radic Biol Med. 104, 298-310. Lin, B., Koibuchi, N., Hasegawa, Y., Sueta, D., Toyama, K., Uekawa, K., Ma, M., Nakagawa, T., Kusaka, H., Kim-Mitsuyama, S., 2014. Glycemic control with empagliflozin, a novel selective SGLT2 inhibitor, ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice. Cardiovasc Diabetol. 13, 148. Li, X., Liu, J., Hu, H., Lu, S., Lu, Q., Quan, N., Rousselle, T., Patel, M. S., and Li, J., 2019. Dichloroacetate ameliorates cardiac dysfunction caused by ischemic insults through AMPK signal pathway- not only shifts metabolism. Toxicol Sci. 167, 604-617. Ma, H., Wang, J., Thomas, D. P., Tong, C., Leng, L., Wang, W., Merk, M., Zierow, S., Bernhagen, J., Ren, J., Bucala, R., Li, J., 2010. Impaired macrophage migration inhibitory factor-AMP-activated protein kinase activation and ischemic recovery in the senescent heart. Circulation 122, 282-292. Mancini, S. J., Boyd, D., Katwan, O. J., Strembitska, A., Almabrouk, T. A., Kennedy, S., Palmer, T. M., Salt, I. P., 2018. Canagliflozin inhibits interleukin-1beta-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms. Sci Rep. 8, 5276. Ma, Y., Li, J., 2015. Metabolic shifts during aging and pathology. Compr Physiol. 5, 667-686. Mazzone, T., 2010. Intensive glucose lowering and cardiovascular disease prevention in diabetes: reconciling the recent clinical trial data. Circulation 122, 2201-2211. Miller, E. J., Li, J., Leng, L., McDonald, C., Atsumi, T., Bucala, R., Young, L. H., 2008. Macrophage migration inhibitory factor stimulates AMP-activated protein 22

kinase in the ischaemic heart. Nature 451, 578-582. Morrison, A., Chen, L., Wang, J., Zhang, M., Yang, H., Ma, Y., Budanov, A., Lee, J. H., Karin, M., Li, J., 2015. Sestrin2 promotes LKB1-mediated AMPK activation in the ischemic heart. FASEB J. 29, 408-417. Morrison, A., Yan, X., Tong, C., Li, J., 2011. Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice. Am J Physiol Heart Circ Physiol. 301, H895-H902.

Figure Legends Figure 1. EMPA treatment triggers phosphorylation of LKB1, AMPK, ACC and accumulation of PGC-1α in the isolated cardiomyocytes from mouse hearts in a time-dependent manner under normoxia state. (A) Representative immunoblotting of p-LKB1 (Ser428), LKB1, p-AMPK (Thr172), AMPKα, p-ACC (Ser79), ACC, PGC-1α and GAPDH; (B) Bar graph shows relative units of p-LKB1 to LKB1; (C) Bar graph shows relative units of p-AMPK to AMPKα; (D) Bar graph shows relative units of p-ACC to ACC; (E) Bar graph shows relative units of PGC-1α to loading control of GAPDH. Values are means ± SEM, n=8-10, *p＜0.05 vs. Vehicle, respectively. Figure 2. EMPA activates LKB1/AMPK signaling pathway in isolated mice cardiomyocytes under H/R conditions. (A) Representative immunoblotting of p-LKB1 (Ser428), LKB1, p-AMPK (Thr172), AMPKα, p-ACC (Ser79), ACC, PGC-1α and GAPDH; (B) Bar graph shows relative units of p-LKB1 to LKB1; (C) Bar graph shows relative units of p-AMPK to AMPKα; (D) Bar graph shows relative units of p-ACC to ACC; (E) Bar graph shows relative units of PGC-1α to loading control of GAPDH. Values are means ± SEM, n=5, *p＜0.05 vs. Vehicle, respectively; †p<0.05 vs. Normoxia Vehicle, respectively; ‡p<0.05 vs. Normoxia EMPA, respectively; #p<0.05 vs. H/R EMPA without Comp C, respectively. Figure 3. The contractile functions of isolated cardiomyocytes from mice hearts treated with vehicle or EMPA under normoxia or H/R conditions. (A) The representative traces of cell contraction by time with a stimulation at a frequency of 1 Hz were shown under normoxia or H/R conditions.(B)The resting sarcomere length; (C) The maximum velocity of shortening (-dL/dt); (D) The maximum contraction amplitude. Values are means ± SEM, n=40-80 cells per group derived from 3 to 4 mice. *p<0.05 vs. Vehicle, respectively; †p<0.05 vs. Normoxia Vehicle, respectively; 23

‡p<0.05 vs. H/R EMPA without Comp C, respectively. Figure 4. EMPA treatment reduces the generation of mitochondrial superoxide in isolated cardiomyocytes during H/R conditions. (A) Representative images of mitochondrial superoxide in cardiomyocytes from mouse hearts under normixa or H/R conditions. (B)Quantitative analysis of the relative levels of mitochondrial superoxide in cardiomyocytes under normxia or H/R conditions. Values are means ± SEM, n=30-50 cells per group derived from 4 mice, *p<0.05 vs.Vehicle; †p<0.05 vs.Normoxia Vehice; ‡p<0.05 vs. H/R Vehicle without Comp C; #p<0.05 vs. H/R EMPA without Comp C. Figure 5. EMPA ameliorates post-ischemic cardiac dysfunction. More higher heart RPP of EMPA group than that of vehicle group during I/R stress condition (A) and less MI area in EMPA group than that in vehicle group by TTC staining after I/R stress condition (B). No significant differences of RPP between Compound C and Compound C with EMPA group (C) and no significant differences of MI area between Compound C and Compound C with EMPA group after I/R stress condition (D). (n=4 hearts for each group. Mean ± SEM, *p<0.05 vs. vehicle controls). Figure 6. EMPA treatment improves cardiac function during I/R stress condition by echocardiography (A) as shown by EF (B) and FS, with no significant differences of FS among six groups (C). Compound C pre-treatment abolishes the benefical effects of EMPA on ischemic hearts. Values are means ± SEM, n=4, *p<0.05 vs. Vehicle respectively; †p<0.05 vs. Sham Vehicle, respectively; ‡p<0.05 vs. I/R EMPA without Comp C. Figure 7. EMPA treatment reduces MI area caused by I/R stress. (A) Representative sections of the extent of myocardial infarction. (B) Ratio of the area to the area at risk (AAR) to the total myocardial area. (C) Ratio of the infarcted area (INF) to the AAR. Values are means ± SEM, n=4, *p<0.05 vs. Vehicle; †p<0.05 vs. Control Vehicle; ‡p<0.05 vs. Control EMPA. Figure 8. The representative slides are stained with hematoxylin and eosin, demonstrating that EMPA alleviate infiltration of inflammatory cells after I/R stress in vivo. Compound C pre-treatment abolished the beneficial effects of EMPA on inflammatory response during this condition. The yellow arrows represents infiltration of inflammatory cells (mainly neutrophils) in the interstitial space of myocardial tissue near the epicardium.

24

Empagliflozin Attenuates Ischemia and Reperfusion Injury Through LKB1/AMPK Signaling Pathway

Qingguo Lua,b, Jia Liub,c, Xuan Lib, Xiaodong Sunb, Jingwen Zhangb,c, Di Renb,c, Nanwei Tonga, Ji Lib,c,* a

Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, 610041; bDepartment of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS 39216; cDepartment of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612

Short Title: Empagliflozin and Cardiac Ischemic Insult

*

Corresponding to: Ji Li, Ph.D., Department of Surgery, Morsani College of Medicine,

University of South Florida, 12901 Bruce B. Downs Blvd, MDC 110, Tampa, FL 33612. Tel: (813) 974-4917, Email: [email protected]

1

Abstract

The beneficial effects of empagliflozin (EMPA) on cardiac functions during ischemia and reperfusion were characterized. The contractile functions of isolated cardiomyocytes from adult C57BL/6J mice were determined with IonOptix SoftEdgeMyoCam system. The mitochondrial superoxide production was measured by MitoSOX fluorescent probe. The ex vivo isolated heart perfusion system was used to determine the pharmacological effects of EMPA on heart’s contractile functions under both physiological and pathological conditions. The in vivo regional myocardial ischemia and reperfusion by ligation of left artery coronary artery descending (LAD) was used to measure the myocardial infarction caused by ischemia and reperfusion with or without EMPA treatment. The results demonstrated that EMPA treatment significantly improves cardiomyocyte contractility under hypoxia conditions and augments the post-ischemic recovery in the ex vivo heart perfusion system. Furthermore, the in vivo myocardial infarction measurement shows that EMPA treatment significantly reduce myocardial infarct size caused by ischemia and reperfusion. The biochemical analysis demonstrated that EMPA can trigger cardiac AMPK signaling pathway and attenuate mitochondrial superoxide production under hypoxia and reoxygenation conditions. In conclusion, EMPA can trigger AMPK signaling pathways and modulate myocardial contractility and reduce myocardial infarct size caused by ischemia and reperfusion independent of hypoglycemic effect. The results for the first time demonstrate that the activation of AMPK by EMPA could one reason about EMPA’s beneficial effects on heart disease.

Key words: Empagliflozin, Ischemia (hypoxia)/reperfusion injury, Non-diabetic mice, AMPK

2

1.

Introduction

Diabetes is considered as a significant risk factor for cardiovascular disease (CVD), the leading cause of global mortality(Mazzone, 2010,Rawshani, Sattar, Franzen et al., 2018). Aims of diabetes treatment are multiple risk factors control, such as hyperglycemia, hyperlipidemia and hypertension, etc. At present, many kinds of hypoglycemic drugs are available for clinical use. As a request by the US Food and Drug Administration (FDA), clinical research evidence of cardiovascular (CV) safety must be submitted by the pharmaceutical industry before the approval of a novel antidiabetic agent (FDA, 2008). Sodium-glucose cotransporter 2 (SGLT2) inhibitors are new class of antidiabetic agents which act by selectively inhibiting SGLT2 of the renal proximal tubule, with a consequent of increase in urinary excretion of glucose, reducing blood glucose concentration independent of insulin. Canagliflozin, dapagliflozin, empagliflozin (EMPA) and ertugliflozin of this family have been approved by FDA. Encouraging evidence of CV protection effects from several large clinical studies of SGLT2 inhibitors have been published(Zinman, Wanner, Lachin et al., 2015,Kosiborod, Cavender, Fu et al., 2017,Neal, Perkovic, Mahaffey et al., 2017,Wiviott, Raz, Bonaca et al., 2018). EMPA REG OUTCOME (Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes)is the first study for CV protection, which suggested that EMPA exerts a 38% risk reduction in death from CV causes, 32% risk reduction death from any cause, and 35% reduction on risk of hospitalization for HF(Zinman et al., 2015,Kosiborod et al., 2017,Neal et al., 2017,Wiviott et al., 2018). It is noteworthy that this effect of EMPA is independent of its hypoglycemic effect. A new indication has been approved by FDA for EMPA to reduce the risk of CVD death in adult patients with T2DM and CVD in 2016 (FDA, 2016). At present, many studies are trying to explore the mechanism of CV protection of SGLT2 inhibitors independent of hypoglycemia effect. Most of the research are focusing on energy metabolism, inflammation, oxidative stress, myocardial fibrosis and electrolyte homeostasis (Bertero, Prates Roma, Ameri et al., 2018). Multiple studies had been reported that EMPA could improve myocardial fibrosis in obese and 3

diabetic mice (Lee, Chang and Lin, 2017,Ye, Bajaj, Yang et al., 2017) and also play the role of anti-oxidative stress and anti-apoptosis (Lin, Koibuchi, Hasegawa et al., 2014,Zhou and Wu, 2017). Meanwhile, EMPA was found to decrease the infarct area under ischemia/reperfusion (I/R) stress (Andreadou, Efentakis, Balafas et al., 2017) and improve diastolic function of the left ventricle (LV) in diabetic mice (Hammoudi, Jeong, Singh et al., 2017). Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an important serine and threonine protein kinase, which plays an important role in cell energy balance(Hardie, 2004). Increasing the ratio between intracellular adenosine monophosphate (AMP) and adenosine triphosphate (ATP) will activate AMPK by phosphorylation(Hardie and Carling, 1997). In addition, liver kinase B1 (LKB1), calmodulin-dependent protein kinase kinase β (CaMKKβ), and AMPK kinase (AMPKK) are all upstream activators. AMPK also plays an important role in reducing oxidative stress, regulating autophagy, and anti-apoptosis of cardiomyocytes(Bertrand, Ginion, Beauloye et al., 2006,He, Zhu, Li et al., 2013). It has been reported that EMPA can protect the heart by activating AMPK in cardiac microvascular endothelial cells (CMEC) of diabetic mice (Zhou, Wang, Zhu et al., 2018). Canagliflozin can regulate energy metabolism via AMPK activation by inhibiting Complex I in the respiratory chain in HEK-293 cells and hepatocytes (Hawley, Ford, Smith et al., 2016). The basic research on SGLT2 inhibitors was mostly focused on animal models of diabetes and/or obesity. To date, it is unclear if SGLT2 inhibitors could offer protection against hypoxia/reoxygenation (H/R) or I/R injury in non-diabetic and non-obese model through AMPK signaling pathway. This study aims to explore whether short-term treatment of EMPA could protect against H/R or I/R injury in healthy young heart. In this study, we selected healthy young male mice as research objects, explored whether short-term administration of EMPA displays protective effect on cardiomyocytes and cardiac function, and the influence of myocardial infarction (MI) area in vitro and in vivo under H/R and I/R stress conditions. At the same time, we tried to further explore whether AMPK related signaling pathway was involved in this mechanism. 4

2. Materials and Methods Young (12weeks) male C57BL/6J mice were obtained from Jackson Laboratory. EMPA and compound C were purchased from Sigma-Aldrich and ApexBio respectively. All animal activity protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center in this study.

2.1. Cardiomyocytes isolation from mice Heparin IV (Fresenius Kabi) for anticoagulation (Ma and Li, 2015,Li, Liu, Hu et al., 2018)was given by intraperitoneal injection with 1000 units/kg 10 min before the experiment. Mice were killed after anaesthesia with 2%–3% isoflurane and 100% O2. The hearts of mice were excised, then cannulated by aorta and connected to the cardiomyocyte perfusion apparatus (Radnoti). The heart was perfused at 37℃ with a Ca2+ free based buffer (pH 7.2) containing: 135 mM NaCl, 4 mM KCl, 1mM MgCl2, 10 mM HEPES, 0.33 mM NaH2PO4, 10 mM glucose, 10mM 2, 3-butanedione monoxime (Sigma), and 5 mM taurine(Sigma) that was bubbled with 95% O2 and 5% CO2.The heart was digested in 25 ml perfusion buffer with 0.3 mg/g body weight collagenase D (Roche), 0.4 mg/g body weight collagenase B (Roche), and 0.05 mg/g body weight protease type XIV (Sigma) dissolved after 3–5 min of recycling. After digested completely, the heart was removed, torn with tweezers and blown gently, then filtered to get the isolated cardiomyocytes.

2.2. Time gradient experiment of cardiomyocytes under normoxia condition Isolated cardiomyocytes were divided into two groups with vehicle (PBS:DMSO=105:1) and EMPA of 0.5µM respectively. Each group was further divided into five subgroups, which were cultured at a time gradient of 10 min for 50 min under normoxia condition. Samples of cardiomyocytes were harvested at every time points for detecting the level of related signal pathway molecular.

Treatment of cardiomyocytes with hypoxia and reoxygenation 5

Isolated cardiomyocytes were treated with vehicle or 0.5µM EMPA and placed in a nitrogen chamber containing 95%N2 and 5%CO2 for 20min to induce hypoxia. Then they were transfered from the nitrogen chamber to normoxia incubator and allowed to reoxygenate for 20 min. The control groups of cardiomyocytes under normoxia condition were treated with vehicle or 0.5µM EMPA and placed in normal incubator for the same time.

2.3. Groups and drug administration of cardiomyocytes in H/R experiment Isolated cardiomyocytes of mice were randomly divided into six groups according to treatment and conditions (normoxia or H/R): baseline with vehicle (B+V), baseline with EMPA (B+E), H/R with vehicle (H/R+V), H/R with EMPA (H/R+E), H/R with Compound C (H/R+C), H/R with Compound C and EMPA (H/R+C+E). The concentration of EMPA and Compound C were 0.5µM and 20µM respectively according to pharmacokinetics and references (Andreadou et al., 2017,Panchapakesan, Pegg, Gross et al., 2013,Tahara, Takasu, Yokono et al., 2016).

2.4. Immunoblotting The way of immunoblotting was previously described in our publications (Li et al., 2018,Quan, Sun, Wang et al., 2017). Briefly, total protein was extracted by lysis buffer from the cardiomyocytes samples of time gradient and H/R experiments. Concentration of protein was determined by Bradford dye binding method (Bio-Rad). Target proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Millipore). Primary rabbit antibodies LKB1 (3047s), phosphorylated LKB1 (p-LKB1)(3482s), AMPKα (2532s), phosphorylated AMPKα (p-AMPKα)(2535s), acetyl coenzyme A carboxylase (ACC)(3661s), phosphorylated ACC (p-ACC)(3676s), peroxisome proliferator-activated receptor co-stimulator 1α (PGC1α)(3820s) and horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody were purchased from Cell Signaling Technology.

2.5. Contractility measurement of mice cardiomyocytes 6

Extracellular Ca2+ solution was used back to the isolated cardiomyocytes gradually from 0.06 mM to a final concentration of 1.2 mM with the interval incubation time of 15 min for each Ca2+concentration. Then the cells were washed by contraction buffer (10mM glucose,1 mM CaCl2, HEPES buffer to 25ml) for three times. The contractility of cardiomyocytes was measured by SoftEdgeMyocam system (IonOptix Corporation). Cardiomyocytes were placed in a chamber and stimulated with a supra-threshold voltage at a frequency of 1 Hz (Gruntzig, 1978,Moshal, Kumar, Tyagi et al., 2009). The data of changes in sarcomere length, duration of shortening and re-lengthening were recorded and analyzed by IonOptix SoftEdge software. The following parameters can be used to evaluate the cardiomyocytes: resting sarcomere length, the maximum velocity of shortening, maximum contraction amplitude.

2.6. Mitochondrial superoxide measurements of cardiomyocytes Mitochondrial superoxide production, a type of reactive oxygen species (ROS) of cardiomyocytes, was measured by MitoSOX Red (Invitrogen). The cells were loaded with MitoSOX Red (5 µM) for 10 min at 37℃ protected from light, and then washed. Fluorescence microscope (excitation at 514 nm and measuring the emitted light at 585 nm) was employed to obtain the images of cardiomyocytes.

2.7. Isolated heart perfusion (Langendorff) Methods of isolated heart perfusion were described before in our publications(Li et al., 2018,Quan et al., 2017). Briefly, mice were anesthetized with isoflurane (2–3%) and the hearts were excised. After aortic retrograde intubation, isolated hearts were perfused in the Langendorff system at 37°C with Kre bs-Henseleit (K-H) buffer(Morrison, Chen, Wang et al., 2015,Yang, Sun, Quan et al., 2016), which containing: 118 mM NaCl,4.75mM KCl, 0.94mM KH2PO4, 1.2mM MgSO4, 25mM NaHCO3, 1.4mM CaCl2, 7 mM glucose, 0.4 mM oleate, 1% bovine serum albumin (BSA),10 mU/ml insulin. Hearts were perfused for 20 min at a flow speed of 4ml/min, followed by turning off the pump to simulate global ischemia for 40 min or 30 min and then reperfusion for 30 min. A balloon filled by fluid was inserted into the LV cavity and 7

connected to the Chart 8 system (AdInstruments, Colorado Springs) to measure and record heart rate and LV developed pressure, then we calculated the systolic blood pressure and rate-pressure product (RPP). The balloon was inflated to achieve a baseline LV end-diastolic pressure of 5 mmHg that was kept constant during I/R (Morrison et al., 2015,Wang, Tong, Yan et al., 2013).

2.8. Groups and drug administration of isolated heart perfusionexperiment Based on the object of this study, we divided the isolated hearts as four groups: I/R with vehicle (PBS: DMSO = 20000:1) (I/R+V), I/R with EMPA (I/R+E), I/R with Compound C (I/R+C), I/R with Compound C and EMPA (I/R+C+E). EMPA was prepared with K-H buffer at a final concentration of 2.5 µM and perfused into the heart. Compound C was given to the mice by intraperitoneal injection with doses of 1mg/kg body weight one hour before the experiment. Drug administration methods and doses are based on previous studies and pharmacokinetics (Tahara et al., 2016).

2.9. Infarct area measurement after isolated heart perfusion. After 30min of reperfusion, the hearts were removed from the Langendorff system and perfused with 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution by syringe and immersed the whole heart in TTC solution for 10 min. Then the hearts were left in 10% formalin for 48 h and cut into slices about 1 mm thickness and photographed with a Leica microscope (Leica Microsystems). TTC can stain normal myocardial tissue red, while the infarct area can't be stained and appear white. We analyzed infarct area with Image J software (National Institutes of Health)(Li et al., 2018,Morrison, Yan, Tong et al., 2011) and calculate the percentage of infarct size to the whole heart slice for comparation between groups.

2.10.

Ischemia/reperfusion treatment

Mice were anesthetized with 2%–3% isoflurane and placed on a heating pad to keep body warm. A ventilator (Harvard Apparatus) was connected after endotracheal intubation. Left lateral thoracotomy was performed to expose the heart under the 8

operating microscope. We occluded the left anterior descending artery (LAD) by 8–0 nylon suture with a polyethylene tube in the surgical knot to protect the LAD from injury by suture during ligation. ST-segment elevation of electrocardiogram (ECG) and regional blanching of the left ventricle were signs of successful operation. After regional ischemia for 45 min, we closed the chest and suture followed by 24h reperfusion.

2.11.

Groups and drug administration of mice in I/R experiment

The mice were given vehicle, EMPA or Compound C everyday three days before operation. Administration mode of EMPA was gavage with the dose of 10mg/kg body weight. Compound C was given by intraperitoneal injection with the dose of 1mg/kg body weight. Pharmacokinetics and previous literature were consulted for the administration and doses of all drugs (Andreadou et al., 2017,Zhou et al., 2018,Tahara et al., 2016,Cheng, Chen, Li et al., 2016). We divided all the mice as six groups: sham with vehicle (sham+V), sham with EMPA (sham+E), I/R with vehicle (I/R+V), I/R with EMPA (I/R+E), I/R with Compound C (I/R+C), I/R with Compound C and EMPA (I/R+C+E).

2.12.

Echocardiography

After I/R treatment, mice were anesthetized (isoflurane) and transthoracic B-mode echocardiography (Vevo 3100, Visualsonics) were performed to measure cardiac function. We obtain the data of averaged LV ejection fraction (LVEF) and fraction shortening (FS) of all mice by Simpson’s measurements.

2.13.

Infarct size assessment in vivo

Mice were anesthetized and the hearts were excised immediately after 24h of reperfusion. TTC and Evans blue dye were used to stain the hearts, and then they were kept in 10% formalin for 48h. We cut the stained heart into slices of 1mm thickness, and took photos with a Leica microscope (Leica Microsystems). Image J software (National Institutes of Health)(Li et al., 2018,Morrison et al., 2011)was also 9

employed for analysis. The region stained blue from Evans Blue suggested the non-ischemic region (safe area), and the red area stained by TTC represented the ischemic area at risk (AAR). The infarcted area (INF) appeared white because it could not be stained by TTC. Finally, we calculated the proportion of myocardial INF to AAR and carried on statistical analysis.

2.14.

Histological examination

We selected hearts randomly from each groups immediately after reperfusion in vivo. All hearts were excised and washed with PBS to remove residual blood and immersed in 10% formalin overnight, then embedded in paraffin. Hearts from each group were cut into slices of 5µm thickness serially and stained with hematoxylin and eosin stain (H&E) for routine histologic examination (Sun, Quan, Wang et al., 2016,Zhang, Zhao, Quan et al., 2017).

2.15.

Statistical analysis

Data were expressed as means ± standard error of the means (SEM). Two-tailed Student’s t test and one-way ANOVA with Tukey’s test for post hoc comparisons were used by Prism 7.0 (GraphPad Software). p< 0.05 was considered as significant difference.

3. Results

3.1. EMPA activates LKB1/AMPK signaling pathway in isolated mice cardiomyocytes under normoxia and H/R condition We got the samples of 11 subgroups (including 0 min time point) with vehicle or EMPA in time gradient experiment under normoxia state. Western-blot showed that phosphorylated activation of LKB1 and AMPK in EMPA group were more significant than that in vehicle group at corresponding same time point (p< 0.05) (Figs. 1A-C). Although LKB1 in the vehicle group was activated by phosphorylation at 10 and 20 10

min, statistical analysis showed that it was more activated at the same time point in the EMPA group (p< 0.05) (Figs. 1A and B). Phosphorylated LKB1 (p-LKB1) and AMPK (p-AMPK) reached their peak value at 20-30 min of time gradient (Figs. 1B and C). Phosphorylated-ACC (p-ACC) and PGC1α level downstream also increased (Figs. 1D and E) (p< 0.05). Therefore, EMPA can activate LKB1/AMPK and downstream pathways in baseline state. Under the condition of hypoxia for 20 min and reoxygenation for 20 min, we found that LKB1 and AMPK were activated in vehicle group and EMPA group (Figs. 2A-C), the activation of LKB1/AMPK was more significant and lasted longer after EMPA treatment under H/R stress conditions (p< 0.05) (Figs. 2B and C). In H/R+C+E group, AMPK phosphorylation was inhibited by Compound C, however, LKB1 was still significantly activated by EMPA. This further confirms that the activation of AMPK by EMPA is via the upstream molecular-LKB1 in cardiomyocytes of mice. In addition, we also found higher levels of p-ACC and PGC1α in EMPA groups than other groups (p< 0.05) (Figs. 2D and E). Therefore, AMPK can play the roles of protective effect on cardiomyocytes through ACC and PGC1α downstream pathway.

3.2. EMPA improves contractility of isolated cardiomyocytes of mice after H/R stress conditions SoftEdge Myocam system and Ion Wizard software were employed to record and analyze the data of contractile function of cardiomyocytes in H/R experiment (Fig. 3). The results showed that the length of sarcomeres at rest in each group was similar (p> 0.05) (Fig. 3A), indicating that the basal state of all cardiomyocytes was consistent in each group. Under baseline condition, the maximum contraction velocity and amplitude were slightly decreased after treatment of 0.5µM EMPA (p< 0.05). Under H/R stress, the maximal contraction velocity and amplitude of cardiomyocytes were significantly lower than those of baseline state (p< 0.01). But after adding 0.5 µM EMPA to the cardiomyocytes at the same time of H/R stress, the contractile function was improved (p<0.01), but still lower than that of baseline state. Compound C inhibited the activity of AMPK under H/R stress and further damaged the contractile 11

function of isolated cardiomyocytes, which could not be compensated by EMPA (p >0.05) (Figs. 3B and C). These data indicated that EMPA can improve the contractile function of mice cardiomyocytes under H/R stress condition, and AMPK related signal pathway is essential for this effect. We summarized the myocardial sarcomeres shortening of each group in Fig. 3D.

3.3. EMPA reduces mitochondrial superoxide production in isolated cardiomyocytes after H/R stress There were no differences in superoxide production between the vehicle and EMPA group (p> 0.05) under normoxia conditions. Superoxide production increased in H/R group, which can be attenuated by 0.5 µM EMPA (p < 0.05). But they increased significantly after adding Compound C to cardiomyocytes under H/R stress conditions, and EMPA had no significant effect on reducing superoxide in the presence of Compound C(p> 0.05) (Figs. 4A and B). The results indicated that EMPA could reduce the production of superoxide through AMPK pathway, thus exerting the role of anti-oxidant stress under H/R stress conditions.

3.4. EMPA improve the systolic function of heart after I/R in isolated heart perfusion system In the isolated heart perfusion system (Langendorff), vehicle, EMPA and Compound C were perfused to isolated hearts by K-H buffer, respectively. The vehicle and EMPA groups received global ischemia for 40min, then reperfusion for 30min. We found that RPP of EMPA group was significantly higher than that of vehicle group during I/R stress condition (p＜0.05) (Fig. 5A). We had tried to make the isolated hearts ischemia for 40 min in Compound C group as well, unfortunately, the hearts could not regain beating after reperfusion. Therefore, ischemia time of Compound C and Compound C+EMPA groups decreased to 30min, the reperfusion time is still 30min. It was found that there were no significant differences of RPP between the two groups (p> 0.05) under I/R conditions (Fig. 5C). These data show that EMPA could improve the systolic function of isolated heart under I/R condition in the Langendorff system, in 12

which AMPK signaling pathways were involved in this role.

3.5. EMPA reduce the area of myocardial infarction (MI) after I/R in Langendorff system of mice We calculated the percentage of the MI area to the total area of heart in each slice, and compared the proportion of the MI area in each group. The statistical results showed that the percentage of MI area in EMPA group were significantly less than that in vehicle group (p< 0.05) (Figs. 5B). There were no significant differences in the MI area between Compound C and EMPA+Compound C group (p> 0.05) (Figs. 5D). Although the ischemic time of the two groups was shorter, the MI area percentage of them was still larger than that of the vehicle and EMPA groups (p< 0.05). It is suggested that EMPA can reduce the area of MI after I/R in the Langendorff system. When Compound C was used to inhibit AMPK at the same time, EMPA could not play a role in reducing the area of MI, indicating that this role of EMPA was achieved at least partly through the activation of AMPK pathway.

3.6. EMPA improve systolic function of the heart after I/R in vivo Echocardiography was performed before and after LAD surgery (Fig.6A). No significant change in blood glucose was recorded before and after EMPA administration, which excluded the potential hypoglycemic effect of EMPA on cardiac function. LVEF and FS were used as indicators of cardiac systolic function. There was no significant difference in preoperative cardiac systolic function among all groups. LVEF and FS in Sham (Vehicle and EMPA) groups also did not change significantly before and after operation. LVEF and FS in I/R + vehicle group decreased significantly after I/R stress compared with preoperative state, while EMPA can improve LVEF, but not FS, compared with vehicle group under I/R stress (p< 0.05) (Figs.6B and 6C). The decrease of LVEF of mice in I/R + Compound C group was more severe than that in I/R + vehicle group(Fig.6B). EMPA also could not significantly ameliorate the LVEF of mice when they were treated with Compound C under I/R stress (Fig.6B). All the results from echocardiography indicated that EMPA can maintain the systolic function 13

of mice after I/R stress, and this protective effect is largely achieved through AMPK and downstream signal pathways. EMPA can reduce the area of MI after I/R stress in vivo After stained by TTC and Evans blue, we cut the heart into slices of 1mm thickness and took photos (Fig.7A). The proportion of AAR and MI area to the total area of cardiac slices was no statistical differnce in each group (p> 0.05) (Fig.7B), which indicated that the degree of ischemic stress was the same in each group. Statistical analysis results showed that the percentage of MI area in EMPA group was significantly less than that in other three groups (p<0.05) (Fig.7C). There was no difference between Compound C and EMPA+Compound C groups (p>0.05), and the MI area of these two groups was larger than that of vehicle and EMPA groups (p<0.05) (Fig.7C). These results suggested that EMPA can reduce the area of MI in mice after I/R stress in vivo, but it couldn't work when AMPK is inhibited, which indicated that the effect of EMPA is at least partly achieved by activation of AMPK. This is consistent with the results of isolated heart perfusion experiment.

3.7. Histological examination Histological sections and HE staining were performed on the heart tissues of each group (Fig.8). The results showed that there was no significant change in myocardial histology in sham + EMPA group compared with sham + vehicle group. In I/R + vehicle group, there was obvious infiltration of inflammatory cells (most of them are neutrophils) in the interstitial space of myocardial tissue near the epicardium. After EMPA treatment during I/R stress condition, the infiltration of inflammatory cells was alleviated. We could observe more severe infiltration of inflammatory cells in I/R + Compound C group than that in I/R+ vehicle group and EMPA could't improve this pathological change under this condition. In addition, no significant histological changes were observed in cardiomyocytes, then ucleus, cytoplasm and interstitial cells in each group. These results indicated that EMPA may act as an anti-acute myocardial inflammation agent through AMPK pathway under I/R stress. 4. Discussion 14

Ischemic heart disease is the leading cause of death worldwide, and diabetes is an important triggering factor. Therefore, the treatment of diabetes is not simply to control blood glucose, but to control multiple risk factors such as blood pressure, serum lipid, uric acid, etc. in order to reduce the morbidity of CVD, other complications and mortality ultimately. At present, the safety requirements for hypoglycemic drugs is also increasing, at least not to increase the risk of CVD, it would be better if there is CV benefits at the same time. Among the hypoglycemic drugs on market, metformin and glucagon-likepeptide1 (GLP-1) agonists have been proved to have CV protective effects. With the increasing request of FDA on CV risk, more hypoglycemic drugs with CV benefits will appear. SGLT2 inhibitors have been shown to be beneficial in heart failure, ischemic heart disease and all-cause mortality in several large clinical trials(Zinman et al., 2015,Kosiborod et al., 2017,Wiviott et al., 2018). After clinical analysis, the protective effect of SGLT2 inhibitors on heart may be related to lowering blood pressure, weight loss, decreasing serum uric acid level, osmotic diuresis, reducing volume load and hemodynamic changes, etc. Further molecular mechanism is still in the exploratory stage. AMPK is an important energy regulator and protective factor in the heart. Our group and others have shown that AMPK is cardioprotective during ischemia by enhancing glucose uptake and glucose transporter 4 (GLUT4) translocation (Miller, Li, Leng et al., 2008), decreasing apoptosis, improving post-ischemic recovery, and limiting MI(Ma, Wang, Thomas et al., 2010,Russell, Li, Coven et al., 2004). Furthermore, our studies have also demonstrated that pharmacological activation of AMPK by activated protein C could protect the heart against I/R injury(Wang, Yang, Rezaie et al., 2011), and inhibit inflammatory responses during H/R by modulating a JNK-mediated NF-κB pathway(Chen, Li, Zhang et al., 2018). Few studies are focusing on the relation of SGLT2 inhibitors and AMPK now. As mentioned earlier, canagliflozin can activate AMPK in HEK-293 cells and hepatocytes (Hawley et al., 2016). It can also inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK dependent and independent mechanisms (Mancini, Boyd, Katwan et al., 2018). Dapagliflozin could attenuate myocardial inflammation, fibrosis, apoptosis, and 15

diabetic remodeling likely mediated through AMPK activation (Ye et al., 2017). EMPA alleviated diabetic cardiac microvascular endothelial cell (CMEC) injury by inhibiting mitochondrial fission via the activation of AMPK-Drp1 (Dynamin-related protein 1) signaling pathways, preserved cardiac CMEC barrier function through suppressed mitochondrial ROS production and subsequently oxidative stress to impede CMEC senescence (Zhou et al., 2018). So EMPA can be considered as a cardiac microvascular-protection drug to maintain cardiac circulatory function and structure upon hyperglycemic insult. In our study, we found that EMPA can improve the contractile function of isolated cardiomyocytes under H/R conditions and reduce the production of mitochondrial superoxide in vitro. In the Langendorff system, EMPA could increase RPP of isolated hearts and significantly reduce the MI area under I/R stress. Pretreatment with EMPA could significantly improve the LVEF of the mice heart under I/R conditions, and also attenuate the area of MI under this condition in vivo. In this study, there was no significant statistical difference in FS among each group, but the change trend was similar to LVEF, which may be related to the relatively small sample size of this study. Because LVEF is more stereoscopic and accurate by volume measurement, we prefer to use LVEF to represent the systolic function of the heart in this study. The effect of EMPA on reducing infarct size after I/R is very significant and intuitive, which is a very encouraging result. The improvement of cardiac function by EMPA in vitro and in vivo is also closely related to the reduction of MI area. EMPA may also has an anti-acute inflammatory effect under I/R conditions according to pathological section. Therefore, our findings are consistent with the results of the cardioprotective effects of EMPA in clinical studies. Myocardial H/R or I/R injury has been the focus of basic and clinical research. Main mechanisms are associated with increased production of free radicals[including ROS and Reactive Nitrogen Species (RNS)], calcium overload, inflammation and microcirculation disorders. The energy metabolism disorder caused by ischemia and hypoxia leads to the decrease of mitochondrial ATP synthesis, which is an important reason for the above pathophysiological processes. AMPK has been shown to protect 16

myocardial I/R or H/R injury by many studies(Ma et al., 2010,Russell et al., 2004,Wang et al., 2011). Based on our hypothesis, we tried to validate whether AMPK is involved in the cardioprotective effect of EMPA in our study. Western blot was used to detect the expression of the target protein in isolated cardiomyocytes, which avoided the influence of protein expression in other cardiac cells such as vascular endothelial cells and fibroblasts. The results showed that AMPK could be phosphorylated and activated in baseline, H/R and I/R state after EMPA treatment of 0.5 µm. Although ischemia or hypoxia can also activate AMPK, it may not completely compensate for the damage caused by H/R or I/R. AMPK is activated to a higher degree and lasts longer by adding exogenous activator EMPA, thus exerting its cardioprotective effect to a greater extent. To further verify the role of AMPK in cardioprotective effect of EMPA, we used the AMPK inhibitor Compound C. It was found that in the presence of Compound C, EMPA could not play its role in improving cardiomyocytes and cardiac contractile function, reducing superoxide production, attenuating infarct size and anti-acute inflammatory. These results further confirm that the protective effect of EMPA on heart may be achieved through AMPK signaling pathway. As we know, the increase of intracellular AMP/ATP ratio is an important cause of AMPK phosphorylation activation, and LKB1, CaMKKβ and AMPKK are the upstream activating factors of AMPK. In our study, we detected the upstream molecules of AMPK, and found that phosphorylated activation of LKB1 was consistent with that of AMPK, and LKB1 was still activated significantly when Compound C inhibited the activation of AMPK, which indicated that EMPA might activate AMPK through the upstream LKB1. So how does EMPA activate LKB1？Xie et al.(Xie, Dong, Zhang et al., 2006) found that the phosphorylation of LKB1 at Ser428 can enhance the ability of LKB1 to bind and activate AMPK. Protein kinase A (PKA) is the upstream molecule of LKB1, which can phosphorylate LKB1(Huang, Zhu, Chen et al., 2019). Our previous study found that Sirtuin 1 (SIRT1) can increase the deacetylation of LKB1 and increase its phosphorylation(Wang, Quan, Sun et al., 2018). Whether EMPA can directly activate LKB1 by phosphorylation or through PKA, SIRT1 etc. has 17

not been reported. We will carry out further research to reveal this mechanism in the future. At the same time, we detected some target molecules downstream of AMPK by Western blot, the results showed that PGC-1α and phosphorylated ACC levels in EMPA group increased significantly, which was consistent with AMPK activation. We know that AMPK/ACC signaling pathway is an important way to increase fatty acid oxidation and ATP production to improve energy metabolism. AMPK/PGC-1α pathway plays an important role in anti-oxidative stress injury. Energy metabolism disorder and oxidative stress are just important pathophysiological processes of H/R and I/R injury. The reduction of MI area by EMPA can also be explained by AMPK-related signaling pathways to improve myocardial energy metabolism and antioxidant stress, thereby increasing cell survival during I/R stress. Therefore, these results above suggest that the activation of LKB1/AMPK/ACC and LKB1/AMPK/PGC-1a signaling pathways is an important molecular mechanism for the cardioprotective effect of EMPA under H/R and I/R stress. An interesting result was found in cardiomyocyte experiments in vitro. At normoxia condition of baseline, the contractile function of cardiomyocytes decreased after adding 0.5 µM of EMPA compared with that of vehicle group, that is to say, negative inotropic effect was observed. Under H/R condition, the contractile function of cardiomyocytes after treatment of the same concentration of EMPA was stronger than that of H/R with vehicle group but still weaker than baseline. What is the reason? Baartscheer et al(Baartscheer, Schumacher, Wust et al., 2017) found that EMPA could inhibit Na+/H+ exchanger (NHE) in rat and rabbit cardiomyocytes at baseline state, reduce Na+ and Ca2+ in cytoplasm, increase Ca2+ in mitochondria, and show negative inotropic effect. This research can explain the negative inotropic effect of EMPA under baseline condition in our study. Therefore, we speculate that EMPA has a negative inotropic effect on cardiomyocytes mainly by inhibiting NHE at baseline. It can also be interpreted that EMPA may have an "energy-saving" effect under normoxia conditions. Once in H/R and I/R conditions, EMPA mainly increases ATP production through AMPK/ACC pathway, and anti-oxidative stress through AMPK/PGC-1a pathway to achieve myocardial protection and enhancement of 18

contractile function. Therefore, the role of EMPA in balancing energy metabolism under different conditions is vital for maintaining the function of cardiomyocytes. In addition, the most interesting feature of our study is the use of "non-diabetic normal mice model". The results from related clinical trials also indicate that the cardioprotective effect of SGLT2 inhibitors is probably independent of its hypoglycemic effect. Because the clinical indications of SGLT2 inhibitors are type 2 diabetes mellitus, the intervention population of clinical studies are all diabetic patients, and the animal models used in basic research are mostly diabetes or obesity. In our study, isolated cardiomyocyte and heart perfusion experiments in vitro excluded the hypoglycemic effect of EMPA completely, because the hypoglycemic target of EMPA is in the kidney. EMPA administration term is relatively short in vivo, and there is no significant change in blood glucose before and after treatment, which also excludes the effect of hypoglycemic effect. Therefore, the results of this study are important for evaluating the cardioprotective effects and mechanisms of EMPA independent of hypoglycemia. EMPA has been approved for a new indications for reducing CV mortality in type 2 diabetes with CVD (FDA, 2016).. The findings of this study may provide basic research evidence for further expanding the clinical indications of these drugs, and may play a major role in reducing some adverse outcomes of I/R injury in these patients after coronary angioplasty or reconstruction (e.g., coronary artery bypass grafting and percutaneous transluminal coronary angioplasty). At the same time, other studies have reported that EMPA may play a cardioprotective role through other signaling pathways. Zhou et al(Zhou and Wu, 2017) found that EMPA can protect the heart of diabetic rats by inhibiting apoptosis of cardiomyocytes induced by endoplasmic reticulum stress. A study of long-term EMPA treatment to obese mice indicated that EMPA improved myocardial function and reduced infarct size as well as improves redox regulation by decreasing inducible nitric oxide synthase (iNOS) expression and subsequently lipid peroxidation as shown by its surrogate marker malondialdehyde (MDA). The mechanisms of action implicated in the activation of signal transducer and activator of transcription 3 (STAT3) 19

pathway anti-oxidant and anti-inflammatory properties(Andreadou et al., 2017). Some studies suggested that EMPA could not activate AMPK (Andreadou et al., 2017,Hawley et al., 2016). We speculate that the different results may be related to the intervening time or concentration of EMPA and the animal model used. This is a preliminary study on the protective effect and mechanism of EMPA on cardiac H/R and I/R injury in non-diabetic normal mice with some limitations. Firstly, although we found that EMPA could activate LKB1/AMPK signaling pathway in cardiomyocytes of mice, the mechanism by which EMPA activates LKB1 requires further investigation. Secondly, the sample size is relatively small of isolated heart perfusion and in vivo experiments, which needs to be further increased. Thirdly, we only observed the possible anti-acute inflammatory effect of EMPA on pathological slices. Further experiments are needed to determine whether EMPA has this effect and its mechanism. In conclusion, EMPA can improve myocardial contractility and attenuate myocardial infarct size after H/R and I/R stress condition independent of hypoglycemic effect. The cardioprotective effect of EMAP may be achieved by LKB1/AMPK/ACC and LKB1/AMPK/PGC1α signaling pathways.

FUNDING The present study was supported by Sichuan Science and Technology Program [grant number 2019YJ0062], American Diabetes Association [grant number 1-17-IBS-296].

Conflict of Interest The authors declare that they have no conflict of interest.

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p-LKB1 (Ser428), LKB1, p-AMPK (Thr172), AMPKα, p-ACC (Ser79), ACC, PGC-1α and GAPDH; (B) Bar graph shows relative units of p-LKB1 to LKB1; (C) Bar graph shows relative units of p-AMPK to AMPKα; (D) Bar graph shows relative units of p-ACC to ACC; (E) Bar graph shows relative units of PGC-1α to loading control of GAPDH. Values are means ± SEM, n=8-10, *p＜0.05 vs. Vehicle, respectively. Figure 2. EMPA activates LKB1/AMPK signaling pathway in isolated mice cardiomyocytes under H/R conditions. (A) Representative immunoblotting of p-LKB1 (Ser428), LKB1, p-AMPK (Thr172), AMPKα, p-ACC (Ser79), ACC, PGC-1α and GAPDH; (B) Bar graph shows relative units of p-LKB1 to LKB1; (C) Bar graph shows relative units of p-AMPK to AMPKα; (D) Bar graph shows relative units of p-ACC to ACC; (E) Bar graph shows relative units of PGC-1α to loading control of GAPDH. Values are means ± SEM, n=5, *p＜0.05 vs. Vehicle, respectively; †p<0.05 vs. Normoxia Vehicle, respectively; ‡p<0.05 vs. Normoxia EMPA, respectively; #p<0.05 vs. H/R EMPA without Comp C, respectively. Figure 3. The contractile functions of isolated cardiomyocytes from mice hearts treated with vehicle or EMPA under normoxia or H/R conditions. (A) The representative traces of cell contraction by time with a stimulation at a frequency of 1 Hz were shown under normoxia or H/R conditions.(B)The resting sarcomere length; (C) The maximum velocity of shortening (-dL/dt); (D) The maximum contraction amplitude. Values are means ± SEM, n=40-80 cells per group derived from 3 to 4 mice. *p<0.05 vs. Vehicle, respectively; †p<0.05 vs. Normoxia Vehicle, respectively; ‡p<0.05 vs. H/R EMPA without Comp C, respectively. Figure 4. EMPA treatment reduces the generation of mitochondrial superoxide in isolated cardiomyocytes during H/R conditions. (A) Representative images of mitochondrial superoxide in cardiomyocytes from mouse hearts under normixa or H/R conditions. (B)Quantitative analysis of the relative levels of mitochondrial superoxide in cardiomyocytes under normxia or H/R conditions. Values are means ± SEM, n=30-50 cells per group derived from 4 mice, *p<0.05 vs.Vehicle; †p<0.05 vs.Normoxia Vehice; ‡p<0.05 vs. H/R Vehicle without Comp C; #p<0.05 vs. H/R EMPA without Comp C. Figure 5. EMPA ameliorates post-ischemic cardiac dysfunction. More higher heart RPP of EMPA group than that of vehicle group during I/R stress condition (A) and less MI area in EMPA group than that in vehicle group by TTC staining after I/R stress condition (B). No significant differences of RPP between Compound C and Compound C with EMPA group (C) and no significant differences of MI area between 25

Compound C and Compound C with EMPA group after I/R stress condition (D). (n=4 hearts for each group. Mean ± SEM, *p<0.05 vs. vehicle controls). Figure 6. EMPA treatment improves cardiac function during I/R stress condition by echocardiography (A) as shown by EF (B) and FS, with no significant differences of FS among six groups (C). Compound C pre-treatment abolishes the benefical effects of EMPA on ischemic hearts. Values are means ± SEM, n=4, *p<0.05 vs. Vehicle respectively; †p<0.05 vs. Sham Vehicle, respectively; ‡p<0.05 vs. I/R EMPA without Comp C. Figure 7. EMPA treatment reduces MI area caused by I/R stress. (A) Representative sections of the extent of myocardial infarction. (B) Ratio of the area to the area at risk (AAR) to the total myocardial area. (C) Ratio of the infarcted area (INF) to the AAR. Values are means ± SEM, n=4, *p<0.05 vs. Vehicle; †p<0.05 vs. Control Vehicle; ‡p<0.05 vs. Control EMPA. Figure 8. The representative slides are stained with hematoxylin and eosin, demonstrating that EMPA alleviate infiltration of inflammatory cells after I/R stress in vivo. Compound C pre-treatment abolished the beneficial effects of EMPA on inflammatory response during this condition. The yellow arrows represents infiltration of inflammatory cells (mainly neutrophils) in the interstitial space of myocardial tissue near the epicardium.

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Highlights •

Energy sensor AMPK signaling mediates the cardioprotection of Empagliflozin against ischemia and reperfusion injury.

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Empagliflozin treatment augments cardiomyocytes contractile functions during ischemic insults.

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The inflammatory response modulation by Empagliflozin is a critical factor contributing to the beneficial effects on ischemic damage.