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Canadian Journal of Cardiology

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results

Published:August 29, 2019DOI:https://doi.org/10.1016/j.cjca.2019.08.033

      Abstract

      Background

      Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling.

      Methods

      Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction.

      Results

      Empagliflozin significantly attenuated transforming growth factor β1–induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2.

      Conclusions

      We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

      Résumé

      Introduction

      L’empagliflozine, un inhibiteur du SGLT-2, a démontré des réductions notables de la mortalité d’origine cardiovasculaire et des admissions liées à l’insuffisance cardiaque (EMPA-REG OUTCOME). Toutefois, le mécanisme sous-jacent aux effets protecteurs de l’empagliflozine contre l’insuffisance cardiaque demeure encore bien peu connu. Les fibroblastes cardiaques jouent un rôle essentiel dans la progression du remodelage structurel cardiaque et de l’insuffisance cardiaque, en partie, en régulant l’homéostasie de la matrice extracellulaire (MEC). L’objectif de la présente étude était de déterminer si l’empagliflozine a un effet direct sur le remodelage de la MEC à médiation par myofibroblastes cardiaques humains.

      Méthodes

      Nous avons isolé les fibroblastes cardiaques par culture d’explantation de tissus auriculaires humains obtenus lors d’une intervention chirurgicale à cœur ouvert. Nous avons utilisé l’analyse de contraction du gel de collagène pour évaluer l’activité des myofibroblastes. La microscopie confocale nous a permis d’examiner la morphologie cellulaire et le remodelage de la MEC à médiation cellulaire. Nous avons évalué l’expression des marqueurs profibrotiques au moyen de la transcription inverse de la réaction en chaîne par polymérase.

      Résultats

      L’empagliflozine a permis d’atténuer considérablement l’activation des fibroblastes induite par le facteur de croissance transformant β1 par contraction du gel collagène après une exposition de 72 heures à des concentrations croissantes (0,5 μmol/l, 1 μmol/l et 5 μmol/l) qui entraînaient une plus grande atténuation. L’évaluation morphologique a montré que les myofibroblastes exposés à l’empagliflozine étaient de plus petite taille, et avaient des extensions plus courtes et moins nombreuses révélatrices d’un phénotype plus quiescent. De plus, l’empagliflozine a considérablement atténué le remodelage de la MEC à médiation cellulaire selon l’indice d’alignement des fibres de collagène. Le profil de l’expression des gènes a révélé que l’empagliflozine permettait de supprimer de façon significative des marqueurs profibrotiques très importants, dont COL1A1, ACTA2, CTGF, FN1 et MMP-2.

      Conclusions

      Nous proposons de nouvelles données qui montrent l’effet direct de l’empagliflozine sur le phénotype et le fonctionnement des myofibroblastes cardiaques humains par atténuation de l’activité des myofibroblastes et le remodelage du collagène à médiation cellulaire. Ces données fournissent des renseignements très importants sur les effets substantiels de l’empagliflozine selon l’étude EMPA-REG OUTCOME.
      With the advent of new antihyperglycemic agents that have been proven to reduce adverse cardiovascular outcomes, new guides are being developed for their use.
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      • et al.
      A practical guide to the use of glucose-lowering agents with cardiovascular benefit or proven safety.
      ,
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      • et al.
      Diabetes for cardiologists: practical issues in diagnosis and management.
      One of the first to garner major attention is a sodium-glucose cotransporter 2 (SGLT2) inhibitor called empagliflozin (EMPA). Despite the profound benefits of empagliflozin on cardiac mortality and heart failure hospitalizations noted in the BI 10773 (Empagliflozin) Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG OUTCOME) trial,
      • Zinman B.
      • Wanner C.
      • Lachin J.M.
      • et al.
      Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes.
      the underlying mechanism(s) remain elusive. Although various hypotheses have been put forward, they have focused primarily on potentially indirect effects of empagliflozin on either preload reduction (via natriuresis/diuresis), afterload reduction (via reduction in blood pressure), or systemic improvements in myocardial energetics (via increasing glucose and fatty acid oxidation).
      • Verma S.
      • Rawat S.
      • Ho K.L.
      • et al.
      Empagliflozin increases cardiac energy production in diabetes novel translational insights into the heart failure benefits of SGLT2 inhibitors.
      • Lopaschuk G.D.
      • Verma S.
      Empagliflozin’s fuel hypothesis: not so soon.
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      • et al.
      Effects of empagliflozin on blood pressure and markers of arterial stiffness and vascular resistance in patients with type 2 diabetes.
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      SGLT2 Inhibition and cardiovascular events: why did EMPA-REG outcomes surprise and what were the likely mechanisms?.
      Given the modest effects of empagliflozin on cardiometabolic factors (ie, blood pressure, body weight, and glycemic control) and the early onset of treatment effects, some of these hypotheses are thought to be unlikely explanations.
      • Kaul S.
      Is the mortality benefit with empagliflozin in type 2 diabetes mellitus too good to be true?.
      Whether empagliflozin exhibits a direct myocardial effect on critical pathways involved in heart failure remains unclear. Herein, we document a novel and previously unrecognized effect of empagliflozin on extracellular matrix regulation and cell-mediated remodelling by human atrial myofibroblasts. Considering the critical role of myofibroblasts in the development and clinical course of cardiac failure, these data may provide important clues to explain the unprecedented benefit of empagliflozin on heart failure mortality.

      Methods

      For full details of the methods, please see the Supplemental Methods. Here, we provide a condensed description.

      Human cardiac fibroblast isolation and expansion

      With approval from local human research ethics review, cardiac tissue from the right atrial appendage (n = 11) was obtained from patients undergoing routine open heart surgery at the Foothills Medical Center (Calgary, Alberta). Patient characteristics are described in Supplemental Table S1.

      Collagen gel contraction assay

      Collagen gel contraction assay was conducted as previously described.
      • Ngu J.M.C.
      • Teng G.
      • Meijndert H.C.
      • et al.
      Human cardiac fibroblast extracellular matrix remodeling: Dual effects of tissue inhibitor of metalloproteinase-2.

      Cell viability assessment

      Cell viability was assessed with the use of an annexin V–propidium iodide binding assay kit (BD Biosciences, Mississauga, ON) according to the manufacturer’s protocol.

      Assessment of cell morphology and local ECM remodelling

      Cell morphology and local ECM remodelling by individual myofibroblasts were assessed with the use of a noncontractile collagen gel model as previously described.
      • Teng G.
      • Svystonyuk D.
      • Mewhort H.E.M.
      • et al.
      Tetrandrine reverses human cardiac myofibroblast activation and myocardial fibrosis.
      Morphologic parameters included cell area as determined with the use of the Multi-Cell outliner ImageJ plug-in, number of cell extensions, and length of extensions. Local ECM remodelling was quantified by assessment of collagen fibre alignment with the use of confocal reflectance microscopy and ImageJ software with the Oval Profile plug-in. For each patient (n = 3), 4 cells were imaged per treatment group.

      Alpha-smooth muscle actin (α-SMA) expression characterization

      Expression of α-SMA was characterized with the use of flow cytometry as previously described.
      • Teng G.
      • Svystonyuk D.
      • Mewhort H.E.M.
      • et al.
      Tetrandrine reverses human cardiac myofibroblast activation and myocardial fibrosis.

      Western blot

      α-SMA expression and SMAD2/3 phosphorylation was characterized by means of Western blot analysis.

      Reverse-transcription quantitative polymerase chain reaction (RT-qPCR)

      Gene expression levels were normalized to the control group (EMPA 0 μmol/L) for each patient and expressed as a log2 fold change.

      Glucose uptake assessment

      Glucose uptake by myofibroblasts was assessed by measuring the uptake of a fluorescent glucose analogue, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG; Cayman Chemical, Ann Arbor, MI), according to the manufacturer’s protocol.

      Cell proliferation assay

      Cell proliferation of myofibroblasts in response to empagliflozin and varying levels of glucose concentration was assessed with the use of a colorimetric WST-1 assay as a surrogate measure of glycolytic activity (Roche Life Science, Indianapolis, IN).

      Statistical analysis

      Experiments for the data shown (except data from flow cytometry and Western blot) were performed using a minimum of technical triplicates. When more than 2 groups were compared, a 1-way repeated-measures analysis of variance (ANOVA) test was performed followed by a Tukey multiple-comparisons test. For the analysis of the WST-1 assay using 3T3 fibroblasts, a 2-way ANOVA test was performed followed by a Tukey multiple-comparisons test. Student t test was performed for the analysis of fibroblast morphology and collagen fibre alignment. Paired t test was performed for the analysis of α-SMA characterization via flow cytometry and Western blot. Ratio paired t test was performed for the analysis of RT-qPCR data. GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) was used for all statistical analyses, and differences between groups were considered to be statistically significant when P < 0.05. All group data are presented as mean ± SD.

      Results

      Empagliflozin attenuates human atrial myofibroblast activity

      Collagen gel contraction assay is an established in vitro model for wound contraction
      • Ngo P.
      • Ramalingam P.
      • Phillips J.A.
      • Furuta G.T.
      Collagen gel contraction assay.
      • Lijnen P.
      • Petrov V.
      • Fagard R.
      Transforming growth factor-β1–mediated collagen gel contraction by cardiac fibroblasts.
      • Kobayashi T.
      • Liu X.
      • Kim H.J.
      • et al.
      TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels.
      and was performed to assess the functional effects of empagliflozin on human atrial myofibroblast activity. Transforming growth factor (TGF) β1 was used to stimulate myofibroblast activity and induce collagen gel contraction. Gel contraction (%) by TGF-β1 at 72 hours was attenuated by empagliflozin, with increasing concentrations suggesting a dose-dependent inhibitory effect on human atrial myofibroblast activity (Fig. 1, A and B). In addition, the cells were harvested from the collagen gel matrix and cell viability was assessed because cell death could result in decreased gel contraction in addition to decreased activation. Cell viability was assessed by means of annexin V/propidium iodide staining and flow cytometry to measure early- and late-stage apoptosis (Fig. 1C). There were no significant differences between groups for measures of early apoptosis (Fig. 1D) or late apoptosis (Fig. 1E), ensuring that cell toxicity was not a confounding factor in the collagen gel contraction assay.
      Figure thumbnail gr1
      Figure 1Empagliflozin (EMPA) attenuates collagen gel contraction by human atrial myofibroblasts. (A) Representative image of collagen gel contraction by human atrial myofibroblasts at 48 hours after time of release (72-hour total treatment). (B) Collagen gel contraction is represented as a ratio normalized to the control group (EMPA 0 μmol/L = 1). Dose-dependent attenuation of gel contraction was observed with empagliflozin as determined with the use of 1-way repeated-measures analysis of variance (ANOVA) followed by Tukey multiple-comparisons test (n = 11). (C) Flow cytometry results for annexin V and propidium iodide staining of myofibroblasts harvested from cell-ECM constructs after 72-hour treatment. Cell viability was not affected by empagliflozin for all 3 concentrations: There were no significant differences between groups for (D) early apoptosis or (E) late apoptosis as determined by means of 1-way repeated measures ANOVA (n = 3). All data are represented as mean ± SD.

      Empagliflozin attenuates human atrial myofibroblast-mediated ECM remodelling

      We further investigated the effects of empagliflozin on human myofibroblasts with the use of a novel culture platform system. This system comprises a thin noncontractile 3-dimensional (3D) collagen gel matrix supported by a floating nylon grid scaffold to allow direct 3D cell-ECM visualization with the use of confocal microscopy (Fig. 2A). Compared with activated myofibroblasts stimulated by TGF-β1, cells cotreated with empagliflozin exhibited significantly reduced cell size (Fig. 2B), cell extension length (Fig. 2C), and total number of extensions (Fig. 2D), which is indicative of a less activated and more quiescent cell phenotype. Furthermore, collagen fibre alignment was significantly reduced by empagliflozin cotreatment (Fig. 2E) suggesting attenuation of cell-mediated local ECM remodelling. These data provide direct visual evidence that empagliflozin can influence the ECM remodelling capacity of human atrial myofibroblasts.
      Figure thumbnail gr2
      Figure 2Empagliflozin (EMPA) attenuates human atrial myofibroblast activation and local ECM remodelling. (A) Representative confocal microscopy images of human atrial myofibroblasts stained with phalloidin (green) and DAPI (blue) embedded in a 3D collagen matrix (red; autoreflectance) polymerized in a nylod grid. Images were taken after 24-hour treatment. Morphologic parameters were assessed and compared with transforming growth factor β1–stimulated (EMPA 0 μmol/L) myofibroblasts: Cells cotreated with 5 μmol/L empagliflozin exhibited significant reductions in (B) cell size, (C) cell extension length, and (D) number of extensions as determined by means of Student t test. (E) Local extracellular matrix remodelling was also attenuated by empagliflozin, as indicated by significant reductions in collagen fibre alignment. All data are represented as mean ± SD (n = 3).

      Empagliflozin decreases α-SMA expression in human atrial myofibroblasts

      α-SMA is a well established marker of myofibroblasts, and an increase in its expression is a hallmark characteristic of myofibroblast activation.
      • Lijnen P.
      • Petrov V.
      • Fagard R.
      Transforming growth factor-β1–mediated collagen gel contraction by cardiac fibroblasts.
      As such, cells were harvested from the cell-ECM constructs after the collagen gel contraction assay, and their α-SMA expression was characterized with the use of flow cytometry. Cells treated with empagliflozin expressed significantly less α-SMA than the control group (Fig. 3, A-C). Moreover, these data were supported by Western blot analysis: Lower levels of α-SMA protein were found in the cell lysate of the empagliflozin treated group compared with the control group (Fig. 3, D and E). Taken together, these data further suggest an inhibitory effect by empagliflozin on myofibroblast activation. Furthermore, to determine if empagliflozin had any impact on the canonic TGF-β pathway, SMAD2/3 phosphorylation was characterized via Western blot in 3T3 fibroblasts. Empagliflozin did not affect SMAD2/3 phosphorylation (Supplemental Fig. S1).
      Figure thumbnail gr3
      Figure 3Empagliflozin (EMPA) decreases alpha-smooth muscle actin (α-SMA) expression in human atrial myofibroblasts. (A) Characterization of α-SMA expression in human atrial myofibroblasts, harvested from cell-ECM constructs after 72-hour treatment, according to flow cytometry. (B) 5 μmol/L empagliflozin treatment significantly decreased the median fluorescence intensity as determined with the use of paired t test (n = 4). (C) Data also represented as a ratio normalized to the control group (EMPA 0 μmol/L = 1). (D, E) Representative image of Western blot, showing lower α-SMA protein expression (42 kD) in human atrial myofibroblasts treated with 5 μmol/L empagliflozin compared with the control group (EMPA 0 μmol/L); GAPDH (37 kD) was used as a loading control (n = 3). All data are represented as mean ± SD.

      Gene expression in human atrial myofibroblasts

      The gene expression profile of human atrial myofibroblasts was assessed by examining the expression of SGLT2 and key profibrotic markers, including matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs), that are involved in ECM remodelling. The myofibroblasts from the cell-ECM constructs were harvested after the collagen gel contraction assay and RT-qPCR were performed. First, there was no detectable mRNA signal for SGLT2 as determined by means of RT-qPCR. The SGLT2 primers were tested with human kidney samples as positive control to rule out any false-negative signal (data not shown). Second, empagliflozin significantly suppressed expression of ACTA2 (α-SMA), fibronectin (FN1), connective tissue growth factor (CTGF), and collagen (COL1A1 and COL1A2) suggesting antifibrotic effects by empagliflozin (Fig. 4A). Furthermore, there was significant suppression of MMP1, MMP2, and TIMP2 expression levels by empagliflozin, suggesting reduced capacity for ECM turnover (Fig. 4B). Finally, we also examined the effects of empagliflozin on the mRNA expression levels of sodium-hydrogen exchanger (NHE) 1 in a small cohort of patient samples but did not find a statistically significant difference when comparing the empagliflozin-treated atrial myofibroblasts with the control group (Supplemental Fig. S2).
      Figure thumbnail gr4
      Figure 4Empagliflozin (EMPA) suppresses critical profibrotic and ECM remodelling markers. mRNA expression profile of profibrotic markers and matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in human atrial myofibroblasts harvested from cell-ECM constructs after 72-hour empagliflozin treatment, measured with the use of reverse-transcription quantitative polymerase chain reaction. (A) 5 μmol/L empagliflozin treatment significantly suppressed the expression of profibrotic markers, including ACTA2, FN1, CTGF, COL1A1, and COL1A2, as well as (B) MMPs/TIMPs involved in ECM remodelling such as MMP1, MMP2, and TIMP2 as determined with the use of paired-ratio t test. Data are represented as a log2 fold change relative to the control group (EMPA 0 μmol/L) and represented as mean ± SD (n = 5).

      Empagliflozin does not affect glucose uptake in human atrial myofibroblasts

      Because human atrial myofibroblasts do not express SGLT2, we were curious to know whether empagliflozin’s effects were secondary to changes in glucose uptake via other transporters. We performed a glucose uptake assay with the use of human atrial myofibroblasts by measuring the uptake of fluorescent glucose analogue 2-NBDG (Fig. 5). Empagliflozin did not affect the uptake of 2-NBDG compared with the control group, whereas a known glucose transporter (GLUT) 1 inhibitor (apigenin), which served as the positive control, significantly decreased the uptake of 2-NBDG. These data suggest that empagliflozin does not influence glucose uptake and that the effects of empagliflozin on human atrial myofibroblasts are not due to restricted access to glucose.
      Figure thumbnail gr5
      Figure 5Empagliflozin’s (EMPA) effect on glucose uptake by human atrial myofibroblasts. (A) Measurements of fluorescent glucose analogue 2-NBDG uptake by human atrial myofibroblasts at excitation/emission of 485/535 nm. All concentrations of empagliflozin (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) did not have any effect on glucose uptake compared with the control group (EMPA 0 μmol/L). Apigenin (API), a known glucose transporter (GLUT) 1 inhibitor which served as positive control, significantly attenuated 2-NBDG uptake as determined by means of 1-way repeated measures analysis of variance followed by Tukey multiple-comparisons test. (B) Data also represented as a ratio normalized to the control group (EMPA 0 μmol/L = 1). All data are represented as mean ± SD (n = 5).

      Empagliflozin does not affect fibroblast glycolytic activity

      We were also curious if altered glycolytic activity could play a role in empagliflozin’s antifibrotic effect on myofibroblasts. In addition to assessing glucose uptake, we examined the effects of empagliflozin on glycolytic metabolism by measuring WST-1 cleavage products, which are largely dependent on glycolytic NAD(P)H production. TGF-β1–stimulated 3T3 fibroblasts clearly demonstrated increased glycolytic activity with increased glucose concentration: The 450 nm absorbance reading of the WST-1 cleavage product was elevated in higher glucose-concentration groups. However, in all 3 glucose-concentration groups, low (2 mmol/L), normal (5.5 mmol/L), and high (25 mmol/L), there were no differences in the absorbance readings in the empagliflozin-treated groups compared with the control group (EMPA 0 μmol/L), suggesting that it has no effect on glycolytic activity. WZB117, another known GLUT1 inhibitor which served as the positive control, significantly decreased absorbance readings in high- and normal-glucose conditions. None of the cells in the low-glucose condition responded to any of the treatments, which could possibly be due to the cells being metabolically starved and going into a dormant and unresponsive state (n = 4; Fig. 6A). The experiment was repeated with human atrial myofibroblasts under high-glucose condition and similar results were found, with empagliflozin having no significant effect on glycolytic activity (Fig. 6, B and C). Taken together, these data suggest that the effects of empagliflozin on human atrial myofibroblasts may not be due to altered glycolytic activity.
      Figure thumbnail gr6
      Figure 6Empagliflozin’s (EMPA) effect on glycolytic activity in myofibroblasts. (A) Measurement of the cleavage product of WST-1 at 450 nm to evaluate the glycolytic activity of myofibroblasts. transforming growth factor (TGF) β1–stimulated 3T3 fibroblasts (n = 4) displayed higher glycolytic activity with increasing glucose concentrations (low glucose [2 mmol/L, green]; normal glucose [5.5 mmol/L, blue]; high glucose [25 mmol/L, red]). Within each glucose condition, empagliflozin did not affect glycolytic activity, whereas WZB117, a known glucose transporter 1 inhibitor which served as positive control, caused a significant decrease in glycolytic activity in normal- and high-glucose conditions as determined by means of 2-way analysis of variance (ANOVA). *P < 0.05 vs baseline (−TGF-β1); #P < 0.05 vs EMPA 0 μmol/L). (B) Similarly, human atrial myofibroblast glycolytic activity in high-glucose conditions were not affected by empagliflozin, as determined by means of 1-way repeated measures ANOVA (n = 5). (C) Data also represented as a ratio normalized to the control group (EMPA 0 μmol/L = 1). All data are represented as mean ± SD.

      Discussion

      Fibroblasts are the predominant cell type in the human heart that play a primary role in wound healing by regulating ECM composition and turnover.
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      Therefore, interventions that stabilize fibroblast-mediated ECM remodelling are an important therapeutic target for patients with heart failure.
      In the EMPA-REG OUTCOME clinical study, there was a remarkable reduction in cardiovascular mortality (38%) noted in individuals with cardiovascular disease treated with empagliflozin.
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      Interestingly, this was not related to a reduction in either myocardial infarction or stroke, but was driven by a profound and rapid reduction in clinical heart failure. The mechanisms underlying the cardioprotective effects of empagliflozin are matters of considerable discussion and debate. Moreover, the effects of empagliflozin on human cardiac fibroblasts, critical cellular mediators of the progression of heart failure, have not been explored to date.
      In the present report, we provide a novel observation that empagliflozin can directly attenuate cell-mediated ECM remodelling by limiting myofibroblast activation despite stimulation with a profibrotic biopeptide. Using well established in vitro models, we show attenuated ECM remodelling in association with down-regulated ECM gene expression for collagen synthesis and ECM turnover (MMPs and TIMPs). These findings are consistent with, and support, recent nonhuman animal studies that show empagliflozin treatment reducing myocardial interstitial fibrosis as well as structural remodelling,
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      because attenuation of myofibroblast activation would reduce fibrotic remodelling to potentially explain these results. Such an effect is likely to reduce left ventricular mass, improve diastolic function, and reduce cardiac failure biomarkers (A-type and B-type natriuretic peptides), as has been suggested in other recent clinical and experimental studies of empagliflozin.
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      The present data add to the growing list of potential mechanisms that have been postulated. These include improvement of ventricular loading conditions,
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      favourable changes in myocardial metabolism through improving myocardial energetics,
      • Verma S.
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      Empagliflozin increases cardiac energy production in diabetes novel translational insights into the heart failure benefits of SGLT2 inhibitors.
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      Empagliflozin’s fuel hypothesis: not so soon.
      ,
      • Ferrannini E.
      • Mark M.
      • Mayoux E.
      CV protection in the EMPA-REG OUTCOME trial: a thrifty substrate hypothesis.
      and direct inhibition of myocardial sodium-hydrogen exchange.
      • Baartscheer A.
      • Schumacher C.A.
      • Wust R.C.I.
      • et al.
      Empagliflozin decreases myocardial cytoplasmic Na+ through inhibition of the cardiac Na+/H+ exchanger in rats and rabbits.
      Although we found no SGLT2 receptor in human atrial myofibroblasts, similar SGLT2-independent effects of empagliflozin were observed by Baartscheer et al. in rat cardiomyocytes with respect to NHE-1, showing that empagliflozin reduced cytoplasmic sodium and calcium levels.
      • Baartscheer A.
      • Schumacher C.A.
      • Wust R.C.I.
      • et al.
      Empagliflozin decreases myocardial cytoplasmic Na+ through inhibition of the cardiac Na+/H+ exchanger in rats and rabbits.
      Ye et al. observed antifibrotic properties and SGLT2-independent effects by another SGLT2 inhibitor, dapagliflozin, on mouse cardiac fibroblasts, showing that it is able to modulate NHE-1 gene expression and association with heat shock protein via adenosine monophosphate–activated protein kinase activation.
      • Ye Y.
      • Bajaj M.
      • Yang H.-C.
      • Perez-Polo J.R.
      • Birnbaum Y.
      SGLT-2 inhibition with dapagliflozin reduces the activation of the Nlrp3/ASC inflammasome and attenuates the development of diabetic cardiomyopathy in mice with type 2 diabetes. Further augmentation of the effects with saxagliptin, a DPP4 inhibitor.
      ,
      • Ye Y.
      • Jia X.
      • Bajaj M.
      • Birnbaum Y.
      Dapagliflozin attenuates Na+/H+ exchanger-1 in cardiofibroblasts via AMPK activation.
      Therefore, it is entirely plausible that empagliflozin has off-target effects outside of SGLT2-mediated pathways in the myocardium to regulate fibroblast activity. In fact, cardiac fibroblasts are known to express NHE-1,
      • Maly K.
      • Strese K.
      • Kampfer S.
      • et al.
      Critical role of protein kinase C α and calcium in growth factor induced activation of the Na+/H+ exchanger NHE1.
      ,
      • Orlowski J.
      • Grinstein S.
      Diversity of the mammalian sodium/proton exchanger SLC9 gene family.
      and nonhuman animal studies have shown that NHE-1 inhibition reduces cardiac fibrosis and remodelling in heart failure models.
      • Engelhardt S.
      • Hein L.
      • Keller U.
      • Klambt K.
      • Lohse M.J.
      Inhibition of Na+/H+ exchange prevents hypertrophy, fibrosis, and heart failure in β1-adrenergic receptor transgenic mice.
      • Juneman E.B.
      • Arsanjani R.
      • Thai H.M.
      • et al.
      Development of a novel sodium-hydrogen exchanger inhibitor for heart failure.
      • Chen T.I.
      • Tu W.C.
      Exercise attenuates intermittent hypoxia-induced cardiac fibrosis associated with sodium-hydrogen exchanger-1 in rats.
      Furthermore, intracellular calcium is known to play a major role in myofibroblast contractile function and remodelling by binding to calmodulin and facilitating the cross-bridge cycle.
      • Castella L.F.
      • Buscemi L.
      • Godbout C.
      • Meister J.-J.
      • Hinz B.
      A new lock-step mechanism of matrix remodelling based on subcellular contractile events.
      • Godbout C.
      • Follonier Castella L.
      • Smith E.A.
      • et al.
      The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.
      • Raizman J.E.
      • Komljenovic J.
      • Chang R.
      • et al.
      The participation of the Na+-Ca2+ exchanger in primary cardiac myofibroblast migration, contraction, and proliferation.
      • Follonier L.
      • Schaub S.
      • Meister J.-J.
      • Hinz B.
      Myofibroblast communication is controlled by intercellular mechanical coupling.
      Thus, if empagliflozin similarly affects calcium level in cardiac myofibroblasts as it does in cardiomyocytes, as reported by Baartscheer et al., it could potentially explain the attenuation of the myofibroblast-mediated gel contractions and local ECM remodelling documented in this study. Reduced cytoplasmic calcium levels may perhaps interfere with the calcium-calmodulin–mediated myosin light chain kinase activation involved in the contractile mechanics. This is speculative and further investigation exploring this hypothesis is required.

      Limitations

      The study’s limitations should be noted. Human patient samples were used in this study for their translational value. However, there are inherent constraints when using patient specimens, including diverse medication profile, varied genetic and demographic background, and overall medical history. These differences may be reflected in the interpatient variability in the data. Furthermore, these primary cells were passaged up to 6 times to obtain the yield required for the experiments, and studies have shown that cardiac fibroblasts can undergo phenotypic changes, such as increased α-SMA expression and myofibroblast activation, with multiple passages.
      • Zhou Y.
      • Richards A.M.
      • Wang P.
      Characterization and standardization of cultured cardiac fibroblasts for ex vivo models of heart fibrosis and heart ischemia.
      • Santiago J.J.
      • Dangerfield A.L.
      • Rattan S.G.
      • et al.
      Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts.
      • Fu X.
      • Khalil H.
      • Kanisicak O.
      • et al.
      Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.
      These changes may have potentially affected the effects of empagliflozin, especially in the later-passage cells. Despite this, paired analyses showed that the effects of empagliflozin were reasonably consistent. In addition, given the difficulties of obtaining ventricular tissue, fibroblasts were derived from patient atrial appendages. Although not likely, given findings in the literature, this may limit the ability to draw conclusions on ventricular remodelling.
      A potential limitation to this study’s clinical translatability may be the concentrations of empagliflozin examined. The oral therapeutic doses used in the EMPA-REG OUTCOME study were 10 mg and 25 mg daily.
      • Zinman B.
      • Wanner C.
      • Lachin J.M.
      • et al.
      Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes.
      Studies have shown that steady-state dosing at 25 mg/d leads to a peak plasma concentration (Cmax) of ~0.7 μmol/L and can reach 1 μmol/L in patients with renal or hepatic dysfunction.
      • Scheen A.J.
      Pharmacokinetic and pharmacodynamic profile of empagliflozin, a sodium glucose co-transporter 2 inhibitor.
      We examined empagliflozin concentrations up to 5 μmol/L because we were observing its relatively acute effects at 24-72 hours in vitro from a single dose as opposed to its long-term effects with steady-state dosing as in EMPA-REG OUTCOME. Moreover, empagliflozin is 86.2% protein bound,
      • Ndefo U.A.
      • Anidiobi N.O.
      • Basheer E.
      • Eaton A.T.
      Empagliflozin (Jardiance): a novel SGLT2 inhibitor for the treatment of type-2 diabetes.
      and because only free drug can diffuse into tissue and act at target sites, to reach these concentrations it may have to accumulate in lipophilic environments, such as plasma membranes, over time in vivo. Cytotoxicity with these upper concentrations was not a concern, because early clinical safety trials of oral doses of empagliflozin associated with Cmax of up to 8 μmol/L showed them to be completely safe in humans.
      • Seman L.
      • Macha S.
      • Nehmiz G.
      • et al.
      Empagliflozin (BI 10773), a potent and selective SGLT2 inhibitor, induces dose-dependent glucosuria in healthy subjects.
      Other studies have used up to 10 μmol/L empagliflozin without any signs of cytotoxicity,
      • Shi X.
      • Verma S.
      • Yun J.
      • et al.
      Effect of empagliflozin on cardiac biomarkers in a zebrafish model of heart failure: clues to the EMPA-REG OUTCOME trial?.
      and the cell viability assay that we performed (which is indicative of early and late apoptosis) exhibited no toxic effects.
      TGF-β1 was used as a profibrotic agent to activate myofibroblasts in the present study; however, in vivo myofibroblast activation can occur in response to other cytokines or mechanical stress. Therefore, we do not know if empagliflozin would have the same effect when human cardiac myofibroblasts are activated by other profibrotic stimuli. It is plausible that the empagliflozin effects documented in this study were a TGF-β1 dependent. Moreover, empagliflozin was administered simultaneously with TGF-β1 as a cotreatment. Therefore, the data can not address whether empagliflozin can reverse activation or dedifferentiate an activated myofibroblast.
      Finally, this study does not provide a mechanistic explanation for empagliflozin’s antifibrotic effect on human cardiac myofibroblasts, especially considering that these cells lack SGLT2. Future studies should evaluate the direct effects of NHE-1 inhibition on cardiac myofibroblasts to see if it has effects similar to empagliflozin. Also, intracellular calcium levels and contractile mechanics should be examined in cardiac myofibroblasts to determine if empagliflozin alters the mobilization of these cations, thereby affecting contractile functions.

      Conclusion

      We provide novel data supporting a direct effect of empagliflozin on human cardiac myofibroblast-mediated ECM regulation and remodelling. These data suggest that empagliflozin modulates a critical cellular mechanism that underlies the progression of clinical heart failure via a direct effect on profibrotic pathways. Given these novel observations on human cardiac myofibroblasts, it is possible that the beneficial effects of empagliflozin also manifest for conditions of heart failure not associated with diabetes. Indeed, such studies are currently underway (NCT03057977).

      Funding Sources

      This work was supported by the Canadian Institutes of Health Research (S.K., S.V., and P.W.M.F.), the Heart and Stroke Foundation of Canada (S.V. and P.W.M.F.), and Alberta Innovates - Health Solutions (S.K.).

      Disclosures

      The authors have no conflicts of interest to disclose.

      Supplementary Material

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      Linked Article

      • It’s Not All About the Cardiomyocyte: Fibroblasts, Empagliflozin, and Cardiac Remodelling
        Canadian Journal of CardiologyVol. 36Issue 4
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          Diabetes mellitus (DM) continues to increase in prevalence in the developed world and is rapidly affecting developing nations.1 It is estimated that by the year 2040, approximately 642 million adults will have DM.2 Type 2 DM (T2DM) will account for approximately 95% of all cases of DM, far outweighing that of type 1 DM. Of note, T2DM has been closely linked to cardiovascular disease and heart failure (HF).3,4 The Framingham study was the first large-scale study to demonstrate the link between DM and HF.
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