Empagliflozin protects heart from inflammation and energy depletion via AMPK activation

Chintan N. Koyani, Ioanna Plastira, Harald Sourij, Seth Hallstro¨m, Albrecht Schmidt, Peter P. Rainer, Heiko Bugger, Sasˇa Frank, Ernst Malle, Dirk von Lewinski

PII: S1043-6618(20)31178-6
DOI: https://doi.org/10.1016/j.phrs.2020.104870
Reference: YPHRS 104870

To appear in: Pharmacological Research

Received Date: 20 December 2019
Revised Date: 22 April 2020
Accepted Date: 22 April 2020

Please cite this article as: Koyani CN, Plastira I, Sourij H, Hallstro¨m S, Schmidt A, Rainer PP, Bugger H, Frank S, Malle E, von Lewinski D, Empagliflozin protects heart from inflammation and energy depletion via AMPK activation, Pharmacological Research (2020),
doi: https://doi.org/10.1016/j.phrs.2020.104870

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© 2020 Published by Elsevier.

Empagliflozin protects heart from inflammation and energy depletion via AMPK activation

Chintan N. Koyani1,*, Ioanna Plastira2, Harald Sourij3,4, Seth Hallström5, Albrecht Schmidt1, Peter

P. Rainer1, Heiko Bugger1, Saša Frank2, Ernst Malle2, Dirk von Lewinski1*

1Division of Cardiology, Medical University of Graz, Graz, Austria

2Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria
3Department of Internal Medicine, Division of Endocrinology and Diabetology, Medical University of Graz, Graz, Austria
4Center for Biomarker Research in Medicine, Graz, Austria

5Division of Physiological Chemistry, Otto Loewi Research Center, Medical University Graz, Graz, Austria
*Joint corresponding authors.

Word count: abstract – 247; article – 3896 Address correspondence to:
Chintan Koyani, PhD
Division of Cardiology, Medical University of Graz, Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
Tel: +43-316-385-71949
Fax: +43-316-385-13733
E-mail: [email protected]
or
Assoc. Prof. Dr. Dirk von Lewinski, MD, PhD
Division of Cardiology, Medical University of Graz, Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
Tel: +43-316-385- 80684
Fax: +43-316-385-13733
E-mail: [email protected]

Graphical abstract

Abstract Aims
Sodium-glucose co-transporter 2 (SGLT2) were originally developed as kidney-targeting anti- diabetic drugs. However, due to their beneficial cardiac off-target effects (as SGLT2 is not expressed in the heart), these antagonists currently receive intense clinical interest in the context of heart failure (HF) in patients with or without diabetes mellitus (DM). Since the mechanisms by which these beneficial effects are mediated are still unclear yet, inflammation that is present in DM and HF has been proposed as a potential pharmacological intervention strategy. Therefore, we tested the hypothesis that the SGLT2 inhibitor, empagliflozin, displays anti-inflammatory potential along with its glucose-lowering property.

Methods and Results

Lipopolysaccharide (LPS) was used to induce inflammation in vitro and in vivo. In cardiomyocytes and macrophages empagliflozin attenuated LPS-induced TNFα and iNOS expression. Analysis of intracellular signalling pathways suggested that empagliflozin activates AMP kinase (AMPK) in both cell types with or without LPS-treatment. Moreover, the SGLT2 inhibitor increased the expression of anti-inflammatory M2 marker proteins in LPS-treated macrophages. Additionally, empagliflozin-mediated AMPK activation prevented LPS-induced ATP/ADP depletion. In vivo administration of LPS in mice impaired cardiac contractility and aortic endothelial relaxation in response to acetylcholine, whereby co-administration of empagliflozin preserved cardiovascular function. These findings were accompanied by improved cardiac AMPK phosphorylation and ATP/ADP, reduced cardiac iNOS, plasma TNFα and creatine kinase MB levels.
Conclusion

Our data identify a novel cardio protective mechanism of SGLT2 inhibitor, empagliflozin, suggesting that AMPK activation-mediated energy repletion and reduced inflammation contribute to the observed cardiovascular benefits of the drug in HF.

Keywords: Heart failure, Empagliflozin, SGLT2, AMPK, Inflammation, ATP/ADP

1. Introduction

Cardiovascular diseases (CVD) are the leading cause of death worldwide and in 2016, 17.9 million people died from CVD [1]. In particular, heart failure (HF) is an increasing clinical challenge and remains a major cause for morbidity and mortality in patients with type 2 diabetes

mellitus (DM) [2]. In general, these diseases demonstrate systemic/local inflammation, metabolic alterations, deteriorated cardiovascular contractility and electrophysiological dysfunction [2, 3].
In a clinical trial, a new anti-diabetic class of drugs, sodium-glucose co-transporter 2 (SGLT2) inhibitors (gliflozins), have shown surprisingly positive benefits in HF patients with DM [4-6]. Empagliflozin was the first drug of the class to show a tremendous cardiovascular benefit in the EMPA-REG OUTCOME trial; cardiovascular events, cardiovascular mortality and hospitalization for HF were reduced by 14%, 38%, and 35%, respectively [7]. These data were confirmed for dapagliflozin (DECLARE trial) and canagliflozin (CANVAS program) indicating a class effect of SGLT2 inhibitors [5]. Most importantly, the very recent DAPA-HF trial showed that dapagliflozin reduces the risk of worsening HF or cardiovascular mortality in HF patients with reduced ejection fraction (EF) with and without DM [6]. Therefore, SGLT2 inhibitors have gained intense clinical interest in terms of mechanism of action of their beneficial effects in HF.
Since the primary target of gliflozins, SGLT2, is not expressed in the heart but primarily in the kidney, it is intriguing that empagliflozin displays beneficial off-target effects on the cardiovascular system. In addition to SGLT2 inhibition, empagliflozin has been shown to inhibit the Na+/H+ exchanger in cardiomyocytes [8], and to reduce myocardial ketone metabolism [9]. Moreover, in db/db mice empagliflozin administration increased ATP production rate by 31% resulting in an elevated cardiac energy status [10]. Previous studies have shown that SGLT2 inhibitors have a potential to activate AMPK in various cell- and animal-models [11-14].
Of note, DM patients show higher blood levels of lipopolysaccharides (LPS), which is a trigger for endotoxemia [15]. Inflammatory signalling cascades include mitogen-activated protein kinases (MAPKs – p38, ERK, JNK), JAK/STAT, p65 (nuclear factor- κB subunit), Akt, AMP- activated protein kinase (AMPK) and others in various cell types [16]. Furthermore, elevated

circulatory cytokine levels, such as tumor necrosis factor a (TNFα), interleukin (IL)-6 and IL-1β, have been observed in patients with HF and/or DM and targeted therapy with the IL-1 β-antibody canakinumab has significantly improved outcome in cardiac patients (CANTOS-trial) [17, 18]. The cardiovascular system is substantially affected by immune cell activation, elevated circulatory cytokines, suppression of fatty acid and/or glucose oxidation, ATP depletion, elevated reactive nitro-oxidative radicals, impaired Ca2+ homeostasis and ion channel function [2, 3].
Based on these observations, we hypothesized that empagliflozin displays anti- inflammatory and energy replenishing properties that are independent of its anti-hyperglycaemic effects, and investigated role of AMPK. To test this hypothesis, we evaluated the anti- inflammatory pathways affected by empagliflozin in vitro using LPS-treated cardiomyocytes and macrophages. For in vivo relevance, we used an endotoxaemic mouse model to assess cardio- protective effects of empagliflozin.

2. Materials and methods

2.1 Cell culture

HL-1 cardiomyocytes (of mouse atrial origin) were cultured in fibronectin (0.5% [w/v])/gelatin (0.02% [w/v]) coated flasks and maintained in Claycomb medium (Sigma-Aldrich, Vienna, Austria) containing 10% (v/v) FBS (Thermo Fisher, IL, USA), 1 g/l D-glucose, 0.1 mM norepinephrine, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin (Sigma- Aldrich) [19]. RAW264.7 macrophages were cultured in DMEM medium (Thermo Fisher) containing 1 g/l D-glucose, 25 mM HEPES, 10% (v/v) FBS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Both cell lines were kept at 37°C under 5% CO2.

2.2 Incubation protocol

HL-1 cells were treated with indicated (in respective Figures and Figure legends) concentrations of LPS (Sigma-Aldrich) and/or empagliflozin (Adooq Bioscience, CA, USA) for indicated time periods. For some experiments cells were treated with AICAR (Tocris, PA, USA), SBI0206965 (Sigma-Aldrich), compound 991 (Adooq Bioscience) or BML275 (Tocris) at indicated concentration 30 min prior to addition of LPS and empagliflozin.

2.3 Animals

The experimental procedure and number of used animals were approved by the ethics committee of the Federal Ministry of Science, Research and Economy of the Republic of Austria (BMWFW- 66.010/0067-V/3b/2018). The experiments were conducted according to the Directive of the European Parliament and of the Council of September 22, 2010 (2010/63/EU). C57Bl6/N mice (male, 8-12 weeks old, 20-28 g) were administered with LPS (5 mg/kg, i.p.) and/or empagliflozin (5 mg/kg, i.p.).

2.4 Western blot analysis

Cells and tissues were lysed in ice-cold lysis buffer (composition in mM: 50 HEPES, 150 NaCl, 1 EDTA, 10 Na4P2O7, 2 Na3VO4, 10 NaF, 1% [v/v] Triton X-100, 10% [v/v] glycerol, pH 7.4 adjusted with NaOH) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (1x, Thermo Fischer Scientific). Protein lysates were centrifuged at 10,000 rpm (4°C, 10 min) to pellet debris. Protein estimation of cell lysates was performed using BCA protein assay kit (Thermo Fischer Scientific). Proteins were separated by electrophoresis on NuPAGE 4-12% Bis-Tris gel (Thermo Fischer Scientific) and transferred to nitrocellulose membranes (Thermo Fischer Scientific). Membranes were blocked with 5% (w/v) non-fat milk in TBST (Tris-buffered saline containing

Tween 20) (25°C, 2 h) and incubated with primary antibodies (overnight at 4°C). The following primary antibodies were used (diluted in 5% [w/v] BSA-TBST): anti-iNOS antibody (1:500, Abcam, Cambridge, UK – ab204017), anti-pp65 (1:1000, Cell Signalling, Leiden, The Netherlands
- 3033), anti-pERK (1:1000, Cell Signalling – 9106), anti-pJNK (1:1000, Cell Signalling – 9251), anti-pp38 (1:1000, Cell Signalling – 9211), anti-pSTAT1 (1:500, Santa Cruz, Vienna, Austria – sc- 8394), anti-pSTAT3 (1:500, Santa Cruz – sc-8059), anti-pSTAT6 (1:500, Santa Cruz – sc-136019), anti-pAkt S473 (1:1000, Cell Signalling – 4060), anti-pAMPKα (1:1000, Cell Signalling – 2531), anti-AMPKα (1:500, Santa Cruz – sc-74461), anti-SGLT1 (1:1000, Abcam – ab14686), anti- SGLT2 (1:500, Santa Cruz – sc-393350) or anti-GAPDH (1:1000, Santa Cruz – sc-32233). After washing, membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:10,000, Cell Signaling – 7076) or goat anti-rabbit IgG (1:10,000, Cell Signaling – 7074) (25°C for 2 h). Immunoreactive bands were visualized using Immobilon Western HRP Substrate (Sigma-Aldrich) and developed by Bio-Rad ChemiDoc MP Imaging System [20].

2.5 RNA isolation and quantitative real-time PCR (qPCR)

QIAshredder and RNeasy Mini Kit (Qiagen, Manchester, UK) were used to isolate RNA according to the manufacturer’s protocol. After reverse transcription using ProtoScript® First Strand cDNA Synthesis Kit (New England BioLabs, Frankfurt, Germany), gene quantification was performed using Luna® Universal qPCR Master Mix (New England BioLabs). The qPCR protocol was performed by LightCycler 480 system (Roche Diagnostics, Vienna, Austria). Following gene specific primers were used: GAPDH (Mm_GAPDH_1_SG), iNOS (Mm_Nos2_1_SG) and TNFα (Mm_Tnf_1_SG) (Qiagen). Relative gene expression levels were normalized to GAPDH and calculated using ΔΔCT method [21].

2.6 Nitrite, TNFα and creatine kinase MB (CK-MB) measurements

Nitrite and TNFα levels of cell culture medium or plasma were analysed using a Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, Vienna, Austria – 780001) and a Mouse TNF alpha ELISA kit (Abcam – ab100747), respectively. In parallel, plasma CK-MB levels were estimated using an ELISA kit (MyBioSource, CA, USA – MBS705293). All the experiments were performed according to the manufacturer’s instructions.

2.7 ATP and ADP measurements

ATP and ADP levels of cells and tissues were determined using a bioluminescence assay kit (Abcam – ab65313) according to the manufacturer’s instructions.

2.8 Flow cytometry

After treatments cells were collected, blocked using the Ultra V blocker (Thermo Scientific), and incubated with PE anti-CD40 or PE anti-CD206 antibody (1:50, Biolegend, Koblenz, Germany) for 40 min (at 4°C). After washing cells were fixed and expression of each surface marker was measured using a Guava easy Cyte 8 Millipore flow cytometer.

2.9 Echocardiography

Mice were mounted on a heated pad, kept under superficial anaesthesia with isoflurane (1.0-2.5%), and fixed in position with continuous monitoring of heart rate and temperature. Chest hair were depilated and a layer of sonographic coupling gel was applied to the thorax and transthoracic echocardiographic measurements were recorded with a Vevo 3100 High Resolution Imaging

System with a 30-MHz RMV-707B scan head (Visual Sonics, ON, Canada). Left ventricular dimensions were analysed using Vevo 3100 Imaging software.

2.10 Wire myography

Rings (~2 mm length) of thoracic aortas were cut and mounted on a small wire myograph chambers. After activation with high potassium solution and norepinephrine (NE) dose-dependent (1 nM-1 µM) contraction, rings were contracted using NE EC80 doses followed by relaxation induced by cumulative addition of acetylcholine (ACh, 1 nM-10 µM). After washing the rings were contracted using NE EC80 doses followed by sodium nitroprusside (SNP, 1 nM-10 µM)- induced relaxation to examine endothelium-independent relaxation. LabChart 8.0 software was used to record and data analysis.

2.11 Cell viability assay

After treatment HL-1 cardiomyocytes were incubated with MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) (0.5 mg/ml; dissolved in serum-free medium) for 30 min at 37°C. The converted insoluble dark purple coloured dye (formazan) was solubilized with 300 µl acidic isopropanol (0.04 M HCl in absolute isopropanol). Cell viability was assessed by measuring optical densities at emission and correction wavelengths of 570 and 630 nm, respectively, using a microtiter plate reader (Victor Multilabel Counter, Perkin-Elmer, Waltham, MA, USA).

2.12 Statistics

Statistical analyses were performed using IBM SPSS Statistics 23 software. Approximate normal distribution of data was assessed by visual (histograms and normal Q-Q plots) and numerical

investigation (z-value of skewness and kurtosis; p value of Shapiro-Wilk test). After checking homogeneity of variance by Levene’s test, between groups comparisons were evaluated by one- way ANOVA (followed by Tukey’s post-hoc test). P-values ≤ 0.05 were considered statistically significant. All tests were 2-sided.

3. Results

3.1 Empagliflozin ameliorates LPS-induced inflammatory responses in cardiomyocytes

In order to induce a dose-dependent induction of inflammatory response of LPS, HL-1 cardiomyocytes were incubated with 0.2-5 µg/ml LPS. We observed an increase in iNOS mRNA expression in a dose-dependent manner starting from 0.2 µg/ml dose and reaching a plateau at 2 µg/ml (Figure 1A). Based on these results, we used 1 µg/ml LPS dose for further experiments. In parallel, LPS induced iNOS mRNA expression starting from 2 h and reaching a plateau at 6 h (Figure 1B).
Next, cardiomyocytes were treated with increasing concentrations of empagliflozin (0.2-2 µM). Starting from 0.5 µM dose, empagliflozin inhibited LPS-induced iNOS expression (Figure 1C). Since 1 µM empagliflozin showed a prominent effect and this dose is comparable to the Cmax of empagliflozin (0.5 and 1.11 µM at 25 and 50 mg dose, respectively, [22]), this concentration was used for further experiments. Next, we analysed iNOS protein expression and extracellular nitrite levels. Experimental data reveal increased iNOS protein expression and extracellular nitrite levels after LPS treatment, whereby empagliflozin pre-treatment showed preventive effects (Figure 1D). Densitometric evaluation of Western blots is shown in Supplementary Figure 1A. To investigate whether empagliflozin directly affects iNOS expression, we performed qPCR analysis

in the absence of LPS. Empagliflozin did not affect iNOS expression of control cardiomyocytes (Supplementary Figure 2A).
Additionally, we analysed TNFα expression. In a time-dependent manner starting from 2 h, LPS-induced TNFα mRNA expression reaching maximum at 8 h (Figure 1E). Pre-treatment of cardiomyocytes with empagliflozin inhibited LPS-induced TNFα expression (Figure 1E). Moreover, analysis of TNFα levels in the supernatant paralleled the results of TNFα mRNA expression (Figure 1F).
Thus, empagliflozin inhibits LPS-induced inflammatory responses in cardiomyocytes at a clinically relevant dose.

3.2 Empagliflozin activates AMPK in LPS-treated cardiomyocytes

LPS is known to activate various intracellular pathways that foster inflammation. Such cascades include p65, MAPKs, JAK-STAT, Akt and AMPK. Indeed, Western blot analyses show activation of cardiac p65, ERK, JNK, p38 MAPK, STAT1/3/6 and Akt in response to LPS (Figure 2A). However, empagliflozin pre-treatment did not affect these signalling cascades (Figure 2A). In contrast, we observed a time-dependent reduction in AMPK phosphorylation upon LPS treatment (Figure 2B) that was restored in the presence of empagliflozin (Figure 2A). Densitometric evaluation of AMPK Western blots is shown in Supplementary Figure 1B,C. These data reveal that empagliflozin counteracts LPS-mediated AMPK dephosphorylation in cardiomyocytes.
As AMPK plays a crucial role in regulation of inflammation and maintenance of cellular energy levels, we aimed to investigate role of empagliflozin-induced AMPK in inflammation and

cellular energy status. As there is no specific AMPK activator or inhibitor available, we used two different AMPK inhibitors (BML275[23] and SBI0206965[24]) and two AMPK activators (AICAR[25] and compound 991[26]) to perform loss and gain function experiments. The protective effects of empagliflozin against LPS-induced extracellular nitrite and TNFα levels were decreased by both inhibitors and augmented by both activators (Figure 2C,D and Supplementary Figure 2B,C). Moreover, empagliflozin counteracted LPS-impaired cellular ATP/ADP (Figure 2E). This effect of empagliflozin was diminished with AMPK inhibition and enhanced with AMPK activation strongly indicating involvement of AMPK in the protective effects of empagliflozin on cardiac energy status (Figure 2E and Supplementary Figure 2D).
In parallel, we investigated whether empagliflozin has potential to increase AMPK phosphorylation in cardiomyocytes without LPS treatment. Interestingly, we observed an increase in AMPK phosphorylation in response to empagliflozin that reached a plateau at 30 min (Figure 2F). Densitometric evaluation of Western blots is shown in Supplementary Figure 1D. This observation was further supported by an increased ATP/ADP ratio by empagliflozin treatment (Figure 2G). Next we investigated whether the SGLT2 inhibitor alters cell viability. Supplementary Figure 3A shows that irrespective of LPS treatment, empagliflozin did not affect cell viability.
Altogether, these data reveal that activation of AMPK by empagliflozin protects cardiomyocytes from LPS-mediated inflammation and energy depletion, and that empagliflozin stimulates AMPK phosphorylation in cardiomyocytes without LPS treatment.

3.3 Induction of an M2 macrophage phenotype by empagliflozin

Cardiac resident as well as circulatory macrophages play a crucial role in development and progression of cardiovascular diseases [27]. Therefore, macrophages were polarized into M1-like pro-inflammatory state using a low dose of LPS (200 ng/ml, Supplementary Figure 3B). LPS treatment resulted in reduced AMPK phosphorylation, whereby empagliflozin pre-treatment showed protective effect (Figure 3A). Moreover, empagliflozin increased pAMPK expression in macrophages without LPS treatment (Figure 3B). Densitometric evaluation of Western blots is shown in Supplementary Figure 1E,F. Loss or gain of AMPK function is considered as a marker of M1- or M2-like macrophage polarization, respectively.
As macrophages are one of the major sources that generate cytokines and nitrites during inflammation [27], we analysed TNFα secretion and iNOS activity (generating nitric oxide [NO]). Treatment of macrophages with LPS increased secretion/activity of both inflammatory markers (Figure 3C,D), while in empagliflozin-treated cells TNFα production and iNOS activity were significantly lower. Pre-incubation of macrophages with AMPK activators and inhibitors revealed that AMPK underlies the protective effects of empagliflozin (Figure 3C,D and Supplementary Figure 2E,F). Additionally, to assess the polarization status of macrophages, we used CD40 and CD206 as M1 and M2 markers, respectively. As expected, FACS analysis reveal increased CD40- positive and decreased CD206-positive cells after LPS treatment. Empagliflozin pre-treated macrophages showed significantly reduced CD40 and increased CD206 staining (Figure 3E,F).
Our in vitro experiments demonstrate that empagliflozin has potential to interfere with the M1 polarization program in inflammatory macrophages and to induce an anti-inflammatory phenotype.

3.4 Empagliflozin inhibits LPS-induced inflammation and energy depletion in vivo

In this set of experiments we aimed to analyse the protective potential of empagliflozin in vivo. Hearts from LPS-treated mice show a marked decrease in AMPK phosphorylation (Figure 4A) and increased iNOS expression (Figure 4B). Co-administration of empagliflozin prevented mice from LPS-induced detrimental effects (Figure 4A/B). Densitometric evaluation of Western blots is shown in Supplementary Figure 1G,H.
In parallel, we collected blood from these mice and determined plasma TNFα and CK-MB levels. ELISA experiments revealed a massive increase in circulating TNFα and CK-MB levels that were significantly lower in the empagliflozin+LPS treatment group (Figure 4C,D). Septic condition reduced cardiac ATP/ADP ratios in mice, while empagliflozin co-administration ameliorated this effect (Figure 4E).
Altogether, these data emphasize a high protective potential of empagliflozin against LPS- induced inflammation and cardiac energy depletion in vivo.

3.5 Impact of empagliflozin on cardiovascular function in LPS treated mice

As empagliflozin showed therapeutic potential against LPS-induced inflammation and energy depletion in vitro as well as in vivo, we further aimed to evaluate cardiac and vascular function. Echocardiography showed a significant reduction in cardiac EF (Figure 5A) and fractional shortening (FS, Figure 5B) in LPS-treated mice. Empagliflozin co-administration rescued heart function from the detrimental effects of LPS (Figure 5A,B). In parallel, thoracic aortas from these mice were used for wire myography experiments. NE-pre-contracted aortic rings from LPS-treated mice showed an impaired ACh-induced relaxation (Figure 5C). Importantly, the

detrimental effect of LPS could be significantly attenuated by empagliflozin co-administration. (Figure 5C). In contrast to ACh-induced relaxation, SNP-induced relaxation was not affected by LPS (Figure 5D). These results demonstrate detrimental effect of LPS on vascular endothelium but not smooth muscle cells and the capacity of empagliflozin to protect vascular endothelium from detrimental effects of LPS.
To summarize, these data suggest that empagliflozin attenuates LPS-induced cardiovascular damage in vivo.

4. Discussion

This study reveals mechanistic insight into the anti-inflammatory and energy replenishing potential of SGLT-2 inhibitor, empagliflozin, in LPS-treated cardiomyocytes and macrophages in vitro as well as LPS-treated mice in vivo. Under in vivo conditions, empagliflozin showed protective effects against LPS-induced CK-MB and TNFα levels, elevated cardiac iNOS expression and depleted cardiac AMPK phosphorylation as well as reduced ATP/ADP ratio. In parallel, cardiovascular function was preserved in LPS±empagliflozin treated mice, as shown by improved EF and FS, and better relaxation of aortic endothelium in response to ACh. At cellular level, empagliflozin attenuated LPS-induced NO and TNFα production in cardiomyocytes and macrophages. Mechanistically our data suggest that empagliflozin did not target LPS-activated MAPKs, JAK/STAT, p65 or Akt pathways, however, the drug increased AMPK phosphorylation in both cell types with or without LPS treatment. Loss of AMPK activity preceded activation of iNOS and TNFα in both cell types. Additionally, empagliflozin promoted M2 polarization of LPS- stimulated macrophages, resulting in an anti-inflammatory phenotype of the immune cells.

In past, a few anti-diabetic drugs were found to be associated with an increased risk of HF, including thiazolidinediones and dipeptidyl peptidase 4 inhibitors [5, 28]. In contrast, several SGLT2 inhibitors, including empagliflozin, have shown positive results against the risk of HF in patients with DM [6, 7]. The beneficial effects in cardiovascular outcome and mortality were also present in non-diabetic HF patients undergoing dapagliflozin treatment [29], albeit lack of SGLT2 in the heart. On the other hand, SGLT1 (another member of the SGLT family) is expressed in the heart [30]. However, the specificity of empagliflozin for SGLT2 (IC50 – 3.1 nM) over SGLT1 (IC50 – 8.3 µM) rules out the possibility of SGLT1 inhibition at the drug dose used in the present study (1 µM) that is comparable to plasma Cmax of empagliflozin (1.1 µM) followed by a 50 mg dose [22].
A link between inflammation and HF has been supported by increased pro-inflammatory markers, including TNFα and interleukins, in HF with reduced and preserved EF [31]. Clinical and animal studies have provided a correlation of inflammation with DM and HF, whereby elevated circulating inflammatory markers were found in patients with DM [2, 32]. Moreover, elevated pro-inflammatory cytokines have been correlated with cardiac fibrosis, ventricular dilation and mortality in patients with HF [17].
Apart from immunity and inflammation, immune cells play crucial roles in maintenance of metabolism and cell function. As the major tissue resident immune cells, macrophages are important cells in immune-metabolic processes contributing tissue (patho)physiology [27]. During HF and ischemic heart diseases resident cardiac macrophages have been reported to recruit infiltrating macrophages. While resident macrophages promote proliferation and angiogenesis to preserve cardiac function, infiltrating macrophages are detrimental and facilitate inflammation, tissue damage and cardiac dysfunction [33]. Our data provide a strong evidence that empagliflozin

promotes AMPK-mediated anti-inflammatory M2-like polarization of macrophages that in turn may contribute to protective effects of the drug against development and progression of HF. Additionally, dapagliflozin was recently shown to promote M2 macrophage polarization via STAT3 signalling [34].
The maintenance of a high ATP/ADP ratio is crucial for cellular survival [35]. Any cellular stress that either accelerates ATP consumption (e.g. muscle contraction) or hinders ATP production (e.g. ischemia or glucose deprivation) results in reduced ATP/ADP ratio and further AMPK activation. AMPK then promotes energy restoration by increasing ATP production and reducing ATP consumption, resulting in higher ATP/ADP ratio [35, 36]. Moreover, AMPK is a key player in (patho)physiological processes of inflammation. Reduced AMPK activity has been observed in failing human and animal hearts [37]. Additionally, previous studies have linked SGLT2 inhibitors with AMPK activation [11-14]. Canagliflozin activated AMPK in hepatocytes and HEK-293 cells, and inhibited endothelial pro-inflammatory cytokines via AMPK-dependent and -independent pathways [12]. Another study proved SGLT1-independent and AMPK- dependent anti-inflammatory effects of dapagliflozin in cardiofibroblasts isolated from type-2 DM [13]. Moreover, empagliflozin showed AMPK-mediated cardiac microvascular-protection and improved mitochondrial function against hyperglycaemia [14]. The present study provides further evidence of AMPK activation by empagliflozin in vivo and in vitro in healthy and disease conditions, whereby AMPK activation blunted inflammatory processes and replenish energy levels. The drug did not increase intracellular AMPK levels, however, increased its activity by enhancing the phosphorylation. Further studies are required to investigate the direct or indirect impact of SGLT2 inhibitors on AMPK activity.

AMPK activation can be achieved by either drug inhibiting mitochondrial function and ATP production (e.g. metformin), drugs acting as an AMP analogue (e.g. AICAR), drugs binding at AMPK allosteric drug and metabolite (ADaM) site (e.g. A-769662 and compound 991) or others [38, 39]. Despite activation of AMPK by SGLT2 inhibitors, canagliflozin reduced ATP/ADP ratio [12], while empagliflozin increased ATP/ADP ratio [40]. Our present data show restoration of ATP/ADP ratio by empagliflozin against LPS-induced energy depletion. These data are in line with the observed increase in ATP levels by empagliflozin in diabetic mice [10]. Our present data reveal empagliflozin-mediated AMPK activation as an early event (10-60 min) that followed restoration of ATP/ADP ratio in a later phase (8 h). In a previous study, compound 991 did not affect basal ATP/ADP ratio, however, increased the ATP/ADP ratio against electrical stimulation- impaired ATP/ADP ratio in skeletal muscle [26]. Similarly, in the present study empagliflozin did not alter basal iNOS expression, however, blunted LPS-induced iNOS expression in cardiomyocytes.
The role of iNOS has been well examined in humans and animals suffering from DM and HF. Although NO is crucial for a number of physiological processes, in excess amount it generates reactive nitrogen species that contribute to impaired vasorelaxation and cardiac contractility [41]. In the heart, NO reduces β-adrenergic responses, inhibits mitochondrial ATP production and impairs Ca2+ homeostasis [41]. In immune cells, AMPK activation is reported to inhibit iNOS expression, which is in line with our findings [42].
Inflammatory conditions negatively impact cardiac and vascular function in vivo. Treatment of diabetic mice with empagliflozin improved myocardial structure, ameliorated oxidative stress and fibrosis, elevated ATP production via glucose and fatty acid oxidation [10]. In a pressure overload-induced HF mouse model, the drug prevented worsening of cardiac function

[43], while in human HF ventricular trabeculae empagliflozin directly improved diastolic function [44]. Additionally, empagliflozin showed improvement of coronary microvascular function in diabetic mice [45]. Our present data show that empagliflozin treatment improved endothelial function in LPS treated mice exemplified by improved ACh-induced vaso-relaxation. Also, we show that the drug improved cardiac contractility and inflammatory markers in vivo. Altogether, the present data reveal that empagliflozin is an AMPK activator and shows protective potential against inflammation and energy depletion in vitro and in vivo. This study provides additional information on SGLT2-independent AMPK-mediated positive effects of empagliflozin on cardiomyocytes and macrophages as well as endothelium, which may in part explain beneficial effects of SGLT2 inhibitors in HF with or without DM.
Conflict of interests

The authors declare no conflict of interest.

Acknowledgements

We highly acknowledge Prof. Wolfgang Sattler (Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria) for his comments and suggestions on the present study and manuscript. We gratefully acknowledge excellent technical support of technicians Christopher Trummer, Sandra Kickmaier, Margarete Lechleitner and Viktoria Herbst. Part of this study was supported by the Austrian National Bank (OENB 17600).
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Figure 1. Empagliflozin ameliorates LPS-induced inflammation in cardiomyocytes

HL-1 cardiomyocytes were incubated with (A) 0.2-5 µg/ml LPS for 8 h, (B) 1 µg/ml LPS for 1-8 h and (C) 1 µg/ml LPS ± 0.2-2 µM empagliflozin for 8 h to determine iNOS expression (n=9).
Alternatively, HL-1 cardiomyocytes were treated with 1 µg/ml LPS ± 1 µM empagliflozin for 8 h to follow (D, upper panel) extracellular nitrite levels, (D, lower panel) iNOS protein expression and (F) extracellular TNFα levels (n=6). (E) TNFα mRNA expression was determined in HL-1 cardiomyocytes incubated with 1 µg/ml LPS (1-8 h) ± 1 µM empagliflozin (8 h, grey bar) (n=9). All values are expressed as mean ± SEM. *p<0.05 vs. control and #p<0.05 vs. LPS by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 2. Empagliflozin activates AMPK in LPS-treated cardiomyocytes

HL-1 cardiomyocytes were incubated with (A) 1 µg/ml LPS ± 1 µM empagliflozin for 30 min and (B) 1 µg/ml LPS for 5-30 min and (F) 1 µM empagliflozin for 10-60 min to follow expression of indicated proteins. HL-1 cardiomyocytes were incubated with indicated combinations of 1 µg/ml LPS, 1 µM empagliflozin, 1 µM SBI0206965 or 10 µM compound 991 for 8 h to determine (C) extracellular nitrite levels, (D) extracellular TNFα levels, and (E,G) cellular ATP/ADP levels. All values are expressed as mean ± SEM (n=6). *p<0.05 vs. control, #p<0.05 vs. LPS and ǂp<0.05 vs. LPS+Empa by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 3. Induction of an M2 macrophage phenotype by empagliflozin

RAW264.7 macrophages were treated with 200 ng/ml LPS ± 1 µM empagliflozin for (A) 30 min and (E,F) 4 h to follow (A) AMPK phosphorylation (n=6), and (E) CD40 and (F) CD206 expression (n=9). (B) Macrophages were treated with 1 µM empagliflozin for 30 min to follow pAMPK expression (n=6). Alternatively, macrophages were incubated with indicated combinations of 200 ng/ml LPS, 1 µM empagliflozin, 1 µM SBI0206965 or 10 µM compound 991 for 8 h to determine (C) extracellular TNFα and (D) extracellular nitrite levels (n=9). All values are expressed as mean ± SEM. *p<0.05 vs. control and #p<0.05 vs. LPS by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 4. Empagliflozin inhibits LPS-induced inflammation and energy depletion in vivo

C57Bl6 mice were injected with 5 mg/kg LPS ± 5 mg/kg empagliflozin. (A) Cardiac AMPK phosphorylation, (B) cardiac iNOS expression, (C) plasma TNFα and (D) CK-MB levels as well as (E) cardiac ATP/ADP levels were analyzed 8 h after LPS±Empa administration. All values are expressed as mean ± SEM (n=8). *p<0.05 vs. control and #p<0.05 vs. LPS by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 5. Impact of empagliflozin on cardiovascular function in LPS treated mice

C57Bl6 mice were injected with 5 mg/kg LPS ± 5 mg/kg Empa for 8 h. Echocardiographic measurements were performed to analyze (A) ejection fraction (EF) and (B) fractional shortening (FS). Thoracic aortas from the mice were used for wire myography. Aortas were pre-contracted using norepinephrine (EC80 from the dose response curve) to analyze dose-dependent relaxation induced by (C) acetylcholine (ACh) or (D) sodium nitroprusside (SNP). All values are expressed as mean ± SEM (n=8). *p<0.05 vs. control and #p<0.05 vs. LPS by one-way ANOVA followed by Tukey’s post-hoc test.BI 10773