Journal Pre-proof

Direct effects of empagliflozin on extracellular matrix remodeling in human cardiac

myofibroblasts: Novel translational clues to EMPA-REG OUTCOME

Sean Kang, BSc, MSc, Subodh Verma, MD PhD, Ali Fatehi Hassanabad, MD MSc,
Guoqi Teng, PhD, Darrell D. Belke, PhD, Jameson A. Dundas, David G. Guzzardi,
BSc, Daniyil A. Svystonyuk, BSc, Simranjit S. Pattar, BSc, MBT, Daniel S.J. Park,
MSc, Jeannine D. Turnbull, BSc, Henry J. Duff, MD PhD, Lee Anne Tibbles, MD
PhD, Ryan H. Cunnington, PhD, Jason R.B. Dyck, PhD, Paul W.M. Fedak, MD PhD
FRCSC

PII: S0828-282X(19)31192-4
DOI: https://doi.org/10.1016/j.cjca.2019.08.033
Reference: CJCA 3431

To appear in: Canadian Journal of Cardiology

Received Date: 18 April 2019

Revised Date: 24 August 2019
Accepted Date: 25 August 2019

Please cite this article as: Kang S, Verma S, Hassanabad AF, Teng G, Belke DD, Dundas JA, Guzzardi
DG, Svystonyuk DA, Pattar SS, Park DSJ, Turnbull JD, Duff HJ, Tibbles LA, Cunnington RH, Dyck
JRB, Fedak PWM, Direct effects of empagliflozin on extracellular matrix remodeling in human cardiac
myofibroblasts: Novel translational clues to EMPA-REG OUTCOME, Canadian Journal of Cardiology
(2019), doi: https://doi.org/10.1016/j.cjca.2019.08.033.

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Direct effects of empagliflozin on extracellular matrix remodeling in

human cardiac myofibroblasts: Novel translational clues to

EMPA-REG OUTCOME

Sean Kang, BSc, MSc1; Subodh Verma, MD PhD2; Ali Fatehi Hassanabad, MD MSc1; Guoqi
Teng, PhD1; Darrell D. Belke, PhD1; Jameson A. Dundas1; David G. Guzzardi, BSc1; Daniyil A.
Svystonyuk, BSc1; Simranjit S. Pattar, BSc, MBT1; Daniel S.J. Park, MSc1; Jeannine D. Turnbull,
BSc1; Henry J. Duff, MD PhD1; Lee Anne Tibbles, MD PhD3; Ryan H. Cunnington, PhD3; Jason
R.B. Dyck, PhD4; Paul W.M. Fedak, MD PhD FRCSC1

1
Section of Cardiac Surgery, Department of Cardiac Sciences,
Cumming School of Medicine, Libin Cardiovascular Institute of Alberta,
University of Calgary, Calgary, Alberta, Canada
2
Division of Cardiac Surgery, St. Michael’s Hospital,
University of Toronto, Toronto, Ontario, Canada
3
Department of Medicine, Cumming School of Medicine,
University of Calgary, Calgary, Alberta, Canada
4
Cardiovascular Research Centre, Mazankowski Alberta Health Institute,
Department of Pediatrics, Faculty of Medicine and Dentistry,
University of Alberta, Edmonton, Alberta, Canada

Address for correspondence:

Paul W.M. Fedak, MD PhD

C880, 1403-29 Street NW

Calgary, Alberta, Canada, T2N 2T9

Phone: (403) 944-5931, Fax: (403) 270-3715

paul.fedak@gmail.com

Total Word Count: 5286

Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

BRIEF SUMMARY

Given empagliflozin’s heart failure protective effects and cardiac fibroblasts’ integral role in the
progression of heart failure, in part, by regulating extracellular matrix (ECM) homeostasis, we
sought to determine empagliflozin’s direct effects on human cardiac myofibroblast-mediated
ECM remodeling. Empagliflozin exhibited direct anti-fibrotic effects on myofibroblasts by
attenuating TGFβ1-induced activation, ECM remodeling, and expression of pro-fibrotic markers.
Our data provides novel translational clues to explain the clinical benefits of empagliflozin on
cardiac failure and mortality.

Word Count: 74

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

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
remodeling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis.
Thus, the objective of this study was to determine if empagliflozin has a direct effect on human
cardiac myofibroblast-mediated ECM remodeling.

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 remodeling was examined using confocal
microscopy. Gene expression of pro-fibrotic markers was assessed using RT-qPCR.

RESULTS: Empagliflozin significantly attenuated TGFβ1-induced fibroblast activation via

collagen gel contraction after 72-hour exposure with escalating concentrations (0.5µM, 1µM, and
5µM) resulting in greater attenuation. Morphological assessment showed 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 remodeling as measured by collagen fiber alignment index. Gene expression
profiling revealed significant suppression of critical pro-fibrotic 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 remodeling. These data provide critical insights into the profound effects of
empagliflozin as noted in the EMPA-REG OUTCOME study.

Word Count: 250

KEY WORDS: Empagliflozin, Fibroblast, Extracellular Matrix

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

INTRODUCTION

With the advent of new antihyperglycemic agents that have been proven to reduce

adverse cardiovascular outcomes, new guides are being developed for their use.1,2 One of the

first to garner major attention is a sodium-glucose cotransporter 2 (SGLT2) inhibitor called

empagliflozin. Despite the profound benefits of empagliflozin noted on cardiac mortality and

heart failure hospitalizations in the EMPA-REG OUTCOME trial,3 the underlying mechanism(s)

remain elusive. Although various hypotheses have been put forward, these 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).4–8

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.9 Whether empagliflozin exhibits a direct myocardial

effect on critical pathways involved in heart failure remain unclear. Herein, we document a

novel and previously unrecognized effect of empagliflozin on extracellular matrix regulation and

cell-mediated remodeling 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.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

METHODS

For full details of the Methods section, please see the supplementary material. 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

Foothills Medical Center (Calgary, Alberta). Patient characteristics are described in

Supplementary Table S1.

Collagen Gel Contraction Assay

Collagen gel contraction assay was conducted as previously described.10

Cell Viability Assessment

Cell viability was assessed using 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 Remodeling

Cell morphology and local ECM remodeling by individual myofibroblasts were assessed

by utilizing a non-contractile collagen gel model as previously described.11 Morphological

parameters include cell area using the Multi-Cell outliner ImageJ plug-in, number of cell

extensions, and length of extensions. Local ECM remodeling was quantified by assessment of

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

collagen fibre alignment using 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 Expression Characterization

Expression of α-SMA was characterized by flow cytometry as previously described.11

Western Blot

α-SMA expression and SMAD2/3 phosphorylation was characterized by western blot

analysis.

Quantitative Reverse Transcription – Polymerase Chain Reaction (RT-qPCR)

Gene expression levels were normalized to the control group (EMPA 0µM) 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 manufacture’s protocol.

Cell Proliferation Assay

Cell proliferation of myofibroblasts in response to empagliflozin and varying levels of

glucose concentration was assessed using a colorimetric WST-1 assay as a surrogate measure of

glycolytic activity (Roche Life Science, Indianapolis, IN).

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

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 two groups were

compared, a one-way repeated-measures ANOVA test was performed followed by a Tukey’s

multiple comparisons test. For the analysis of the WST-1 assay using 3T3 fibroblasts, a two-way

ANOVA test was performed followed by a Tukey’s multiple comparisons test. Student’s 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 statistically significant when P < 0.05. All group data are presented as

mean ± standard deviation (SD).

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

RESULTS

Empagliflozin Attenuates Human Atrial Myofibroblast Activity

Collagen gel contraction assay is an established in vitro model for wound contraction,12–
14
and was performed to assess the functional effects of empagliflozin on human atrial

myofibroblast activity. 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 (Figure 1A-B). Additionally, the cells were harvested from

the collagen gel matrix and cell viability was assessed as cell death could result in decreased gel

contraction in addition to decreased activation. Cell viability was assessed using annexin

V/propidium iodide staining and flow cytometry to measure early and late stage apoptosis

(Figure 1C). There were no significant differences between groups for measures of early

apoptosis (Figure 1D) or late apoptosis (Figure 1E), ensuring that cell toxicity was not a

confounding factor in the collagen gel contraction assay.

Empagliflozin Attenuates Human Atrial Myofibroblast-Mediated ECM Remodeling

We further interrogated the effects of empagliflozin on human myofibroblasts using a

novel culture platform system. This system comprises of a thin non-contractile 3D collagen gel

matrix that is supported by a floating nylon grid scaffold to allow direct 3D cell-ECM

visualization with confocal microscopy (Figure 2A). Compared to activated myofibroblasts

stimulated by TGF-β1, cells co-treated with empagliflozin exhibited significantly reduced cell

size (Figure 2B), cell extension length (Figure 2C) and total number of extensions (Figure 2D),

which is indicative of a less activated and more quiescent cell phenotype. Furthermore, collagen

8
Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

fibre alignment was significantly reduced by empagliflozin co-treatment (Figure 2E) suggesting

attenuation of cell-mediated local ECM remodeling. These data provide direct visual evidence

that empagliflozin can influence the ECM remodeling capacity of human atrial myofibroblasts.

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.13 As such, cells were harvested from the

cell-ECM constructs after the collagen gel contraction assay and their α-SMA expression was

characterized using flow cytometry. Cells treated with empagliflozin expressed significantly

lower α-SMA than the control group (Figure 3A-C). Moreover, this data was supported by

western blot analysis as lower levels of α-SMA protein was found in the cell lysate of the

empagliflozin treated group compared to the control group (Figure 3D-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 canonical TGF-β pathway

SMAD2/3 phosphorylation was characterized via western blot in 3T3 fibroblasts. Empagliflozin

did not affect SMAD2/3 phosphorylation (Supplementary Figure S1).

Gene Expression in Human Atrial Myofibroblasts

The gene expression profile of human atrial myofibroblasts was assessed by examining

the expression of SGLT2 and key pro-fibrotic markers, including MMP/TIMPs, which are

involved in ECM remodeling. The myofibroblasts from the cell-ECM constructs were harvested

after the collagen gel contraction assay and RT-qPCR was performed. First, there was no

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

detectable mRNA signal for SGLT2 by RT-qPCR. The SGLT2 primers were tested with human

kidney samples as a 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 anti-fibrotic effects by empagliflozin (Figure 4A). Furthermore, there was

significant suppression of MMP1, MMP2, and TIMP2 expression levels by empagliflozin

suggesting reduced capacity for ECM turnover (Figure 4B). Lastly, we also examined the

effects of empagliflozin on the mRNA expression levels of sodium-hydrogen exchanger-1

(NHE-1) in a small cohort of patient samples but did not find a statistically significant difference

when comparing the empagliflozin treated atrial myofibroblasts to the control group

(Supplementary Figure S2).

Empagliflozin Does Not Affect Glucose Uptake in Human Atrial Myofibroblasts

Since 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. Thus, a glucose uptake assay was performed using human atrial myofibroblasts by

measuring the uptake of a fluorescent glucose analogue called 2-NBDG (Figure 5A-B).

Empagliflozin did not affect the uptake of 2-NBDG compared to the control group whereas a

known GLUT1 inhibitor (apigenin), which served as the positive control, significantly decreased

the uptake of 2-NBDG. This data suggests 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.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

Empagliflozin Does Not Affect Fibroblast Glycolytic Activity

We were also curious if altered glycolytic activity could play a role in empagliflozin’s

anti-fibrotic 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

is largely dependent on glycolytic NAD(P)H production. TGF-β1 stimulated 3T3 fibroblasts

clearly demonstrated increased glycolytic activity with increased glucose concentration as the

450nm absorbance reading of the WST-1 cleavage product was elevated in higher glucose

concentration groups. However, in all three glucose concentration groups, low (2mM), normal

(5.5mM), high (25mM), there were no differences in the absorbance readings in the

empagliflozin treated groups compared to the control group (EMPA 0µM), 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; Figure 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 (Figure 6B-C). Taken together, this data

suggests that the effects of empagliflozin on human atrial myofibroblasts may not be due to

altered glycolytic activity.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

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.15,16 When stimulated by pro-

fibrotic pathways, fibroblasts become activated myofibroblasts that secrete ECM proteins and

remodel local ECM networks.15,17 When excessive or prolonged this maladaptive process can

result in myocardial fibrosis and structural cardiac chamber remodeling, a hallmark of clinical

heart failure.18,19 Cardiac fibroblasts regulate ECM turnover by expression of MMPs and their

endogenous inhibitors, TIMPs. Increased activation of the myocardial pro-fibrotic machinery,

and an imbalance between MMPs and TIMPs, disrupts ECM, compromising the structural

integrity of the myocardium with resultant progressive chamber dilatation.17 Excess deposition

of collagen promotes ventricular stiffness and impairs ventricular contraction, leading to both

systolic and diastolic heart failure.20 Therefore, interventions that stabilize fibroblast-mediated

ECM remodeling 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.3 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 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 this report, we provide a novel observation that empagliflozin can directly attenuate

cell-mediated ECM remodeling by limiting myofibroblast activation despite stimulation with a

pro-fibrotic biopeptide. Using well established in vitro models we show attenuated ECM

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

remodeling in association with downregulated ECM gene expression for collagen synthesis and

ECM turnover (MMP and TIMP). These findings are consistent with, and support, recent animal

studies that show empagliflozin treatment reducing myocardial interstitial fibrosis as well as

structural remodeling21–23 as attenuation of myofibroblast activation would reduce fibrotic

remodeling to potentially explain these results. Such an effect is likely to reduce left ventricular

mass, improve diastolic function, and reduce cardiac failure biomarkers (ANP and BNP) as has

been suggested in other recent clinical and experimental studies of empagliflozin.23–25

The present data adds to the growing list of other potential mechanisms that have been

postulated. These include improvement of ventricular loading conditions,8,26,27 favorable

changes in myocardial metabolism through improving myocardial energetics,4,5,7 and direct

inhibition of myocardial sodium-hydrogen exchange (NHE).28 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.28 Ye et al. observed anti-fibrotic

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 (HSP70) via AMPK activation.29,30 Therefore, it is entirely plausible that

empagliflozin has off-target effects outside SGLT2 mediated pathways in the myocardium to

regulate fibroblast activity. In fact, cardiac fibroblasts are known to express NHE-131,32 and

animal studies have shown that NHE-1 inhibition reduces cardiac fibrosis and remodeling in

heart failure models.33–35

Furthermore, intracellular calcium is known to play a major role in myofibroblast

contractile function and remodeling by binding to calmodulin and facilitating the cross-bridge

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

cycle.36–39 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 remodeling

documented in this study. Reduced cytoplasmic calcium levels may perhaps interfere with the

calcium-calmodulin mediated myosin light chain kinase (MLCK) 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

specimen, including diverse medication profile, varied genetic and demographic background,

and overall medical history. These differences may be reflected in the inter-patient variability in

the data. Furthermore, these primary cells were passaged up to six 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.40–42 These changes may have potentially impacted the effects of

empagliflozin especially in the later passage cells. In spite of this, paired analyses showed that

the effects of empagliflozin was reasonably consistent. Additionally, given the difficulties of

obtaining ventricular tissue, fibroblasts were derived from patient atrial appendages. Although

not likely given findings in literature, this may limit the ability to draw conclusions on

ventricular remodeling.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

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 10mg and 25mg daily.3 Studies have shown that steady state dosing at 25mg/day leads to a

peak plasma concentration (Cmax) of about 0.7µM and can reach the order of 1µM in patients

with renal or hepatic dysfunction.43 This study examined empagliflozin concentrations up to

5µM since we were observing its relatively acute effects at 24-72 hours in vitro from a single

dose as opposed to its long term effects using steady state dosing as shown in EMPA-REG

OUTCOME. Moreover, empagliflozin is 86.2% protein-bound44 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 since early clinical safety trials examined oral doses of

empagliflozin associated with Cmax of up to 8µM and was shown to be completely safe in

humans.45 Other studies have used up to 10µM of empagliflozin without any signs of

cytotoxicity25 and the cell viability assay we performed (which is indicative of early and late

apoptosis) exhibited no toxic effects.

TGF-β1 was used as a pro-fibrotic agent to activate myofibroblasts in this study; however,

in vivo myofibroblast activation can occur in response to other cytokines or mechanical stress.

Thus, we do not know if empagliflozin will have the same effect when human cardiac

myofibroblasts are activated by other pro-fibrotic stimuli. It is plausible that the empagliflozin

effects documented in this study was a TGF-β1 dependent effect. Moreover, empagliflozin was

administered simultaneously with TGF-β1 as a co-treatment. Thus, the data can not address

whether empagliflozin can reverse activation or de-differentiate an activated myofibroblast.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

Lastly, this study does not provide a mechanistic explanation for empagliflozin’s anti-

fibrotic effect on human cardiac myofibroblasts, especially since these cells lack SGLT2. Future

studies should evaluate the direct effects of NHE-1 inhibition on cardiac myofibroblasts to see if

it has similar effects 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.

CONCLUSIONS

We provide novel data supporting a direct effect of empagliflozin on human cardiac

myofibroblast-mediated ECM regulation and remodeling. These data suggest that empagliflozin

modulates a critical cellular mechanism that underlies the progression of clinical heart failure via

a direct effect on pro-fibrotic 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 Canadian Institutes of Health Research (SK, SV, and

PWMF); by Heart and Stroke Foundation of Canada (SV and PWMF); and by Alberta Innovates

– Health Solutions (SK).

DISCLOSURES
No conflicts of interest relevant to this article were reported.

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

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FIGURE LEGENDS

Figure 1. Empagliflozin attenuates collagen gel contraction by human atrial myofibroblasts

Representative image of collagen gel contraction by human atrial myofibroblasts at 48 hours

after time of release (72-hour total treatment) (A). Collagen gel contraction is represented as a

ratio normalized to the control group (EMPA 0µM = 1). Dose-dependent attenuation of gel

contraction was observed with empagliflozin as determined by a one-way repeated measures

ANOVA followed by a Tukey’s multiple comparisons test (B; n=11). Flow cytometry results for

annexin V and propidium iodide staining of myofibroblasts harvested from cell-ECM constructs

after 72-hour treatment (C). Cell viability was not affected by empagliflozin for all three

concentrations as there were no significant differences between groups for early apoptosis (D) or

late apoptosis (E) as determined by a one-way repeated measures ANOVA (n=3). All data

represented as mean ± SD.

Figure 2. Empagliflozin attenuates human atrial myofibroblast activation and local ECM

remodeling

Representative confocal microscopy images of human atrial myofibroblasts stained with

phalloidin (green) and DAPI (blue) embedded in a 3D collagen matrix (red; auto-reflectance)

polymerized in a nylod grid (A). Morphological parameters were assessed and compared to

TGF-β1 stimulated (EMPA 0µM) myofibroblasts, cells co-treated with 5µM empagliflozin

exhibited significant reductions in cell size (B), cell extension length (C), and number of

extensions (D) as determined by a Student’s t-test. Local ECM remodeling was also attenuated

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

by empagliflozin as indicated by significant reductions in collagen fiber alignment (E). Images

taken after 24-hour treatment. All data represented as mean ± SD (n=3).

Figure 3. Empagliflozin decreases α-SMA expression in human atrial myofibroblasts

Characterization of α-SMA expression in human atrial myofibroblasts, harvested from cell-ECM

constructs after 72-hour treatment, by flow cytometry (A). 5µM empagliflozin treatment

significantly decreased the median fluorescence intensity as determined by a paired t-test (B;

n=4). Data also represented as a ratio normalized to the control group (EMPA 0µM = 1) (C).

Representative image of western blot showing lower α-SMA protein expression (42kDa) in

human atrial myofibroblasts treated with 5µM empagliflozin compared to the control group

(EMPA 0µM); GAPDH (37kDa) was used as a loading control (D-E; n=3). All data represented

as mean ± SD.

Figure 4. Empagliflozin suppresses critical pro-fibrotic and ECM remodeling markers

mRNA expression profile of pro-fibrotic markers and MMP/TIMPs in human atrial

myofibroblasts harvested from cell-ECM constructs after 72-hour empagliflozin treatment,

measured using RT-qPCR. 5µM empagliflozin treatment significantly suppressed the expression

of pro-fibrotic markers including ACTA2, FN1, CTGF, COL1A1 and COL1A2 (A), as well as

MMP/TIMPs involved in ECM remodeling such as MMP1, MMP2, and TIMP2 (B) as

determined by a paired ratio t-test. Data represented as a log2 fold change relative to the control

group (EMPA 0µM) and represented as mean ± SD (n=5).

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Kang et al. EMPA and Myofibroblast-Mediated ECM Remodeling

Figure 5. Empagliflozin’s effect on glucose uptake by human atrial myofibroblasts

Measurements of 2-NBDG uptake by human atrial myofibroblasts at an excitation/emission of

485/535nm (A). All concentrations of empagliflozin (0.5µM, 1µM, 5µM) did not have any

effect on glucose uptake compared to the control group (EMPA 0µM). Apigenin, a known

GLUT1 inhibitor which served as the positive control, significantly attenuated 2-NBDG uptake

as determined by a one-way repeated measures ANOVA followed by a Tukey’s multiple

comparisons test. Data also represented as a ratio normalized to the control group (EMPA 0µM

= 1) (B). All data represented as mean ± SD (n=5).

Figure 6. Empagliflozin’s effect on glycolytic activity in myofibroblasts

Measurement of the cleavage product of WST-1 at 450nm to evaluate the glycolytic activity of

myofibroblasts. TGF-β1 stimulated 3T3 fibroblasts (n=4) displayed higher glycolytic activity

with increasing glucose concentrations [Low glucose (2mM, green); Normal glucose (5.5mM,

blue); High glucose (25mM, red)]. Within each glucose condition empagliflozin did not affect

glycolytic activity, whereas WZB117, a known GLUT1 inhibitor which served as the positive

control, caused a significant decrease in glycolytic activity in normal and high glucose conditions

as determined by a two-way ANOVA (*P<0.05 vs. baseline (-TGFβ), #P<0.05 vs. EMPA 0µM)

(A). Similarly, human atrial myofibroblast glycolytic activity in high glucose conditions were

not affected by empagliflozin as determined by a one-way repeated measures ANOVA (n=5) (B).

Data also represented as a ratio normalized to the control group (EMPA 0µM = 1) (C). All data

represented as mean ± SD.

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