JACC: CARDIOVASCULAR IMAGING VOL. 14, NO.

2, 2021

ª 2021 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION

PUBLISHED BY ELSEVIER

ORIGINAL RESEARCH

Empagliflozin Ameliorates Diastolic

Dysfunction and Left Ventricular
Fibrosis/Stiffness in Nondiabetic
Heart Failure
A Multimodality Study

Carlos G. Santos-Gallego, MD, Juan Antonio Requena-Ibanez, MD, Rodolfo San Antonio, MD,
Alvaro Garcia-Ropero, MD, Kiyotake Ishikawa, MD, Shin Watanabe, MD, Belen Picatoste, PHD,
Ariana P. Vargas-Delgado, MD, Eduardo J. Flores-Umanzor, MD, Javier Sanz, MD, Valentin Fuster, MD, PHD,
Juan J. Badimon, PHD

ABSTRACT

OBJECTIVES The purpose of this study was to investigate the effect of empagliflozin on diastolic function in a
nondiabetic heart failure with reduced ejection fraction (HFrEF) scenario and on the pathways causing diastolic
dysfunction.

BACKGROUND This group demonstrated that empagliflozin ameliorates adverse cardiac remodeling, enhances
myocardial energetics, and improves left ventricular systolic function in a nondiabetic porcine model of HF. Whether
empagliflozin also improves diastolic function remains unknown. Hypothetically, empagliflozin would improve diastolic
function in HF mediated both by a reduction in interstitial myocardial fibrosis and an improvement in cardiomyocyte
stiffness (titin phosphorylation).

METHODS HF was induced in nondiabetic pigs by 2-h balloon occlusion of proximal left anterior descending artery.
Animals were randomized to empagliflozin or placebo for 2 months. Cardiac function was evaluated with cardiac mag-
netic resonance (CMR), 3-dimensional echocardiography, and invasive hemodynamics. In vitro relaxation of cardiomyo-
cytes was studied in primary culture. Myocardial samples were obtained for histological and molecular evaluation.
Myocardial metabolite consumption was analyzed by simultaneous blood sampling from coronary artery and coronary
sinus.

RESULTS Despite similar initial ischemic myocardial injury, the empagliflozin group showed significantly improved dia-
stolic function at 2 months, assessed by conventional echocardiography (higher e0 and color M-mode propagation velocity,
lower E/e0 ratio, myocardial performance Tei index, isovolumic relaxation time, and left atrial size), echocardiography-
derived strain imaging (strain imaging diastolic index, strain rate at isovolumic relaxation time and during early diastole, and
untwisting), and CMR (higher peak filling rate, larger first filling volume). Invasive hemodynamics confirmed improved
diastolic function with empagliflozin (better peak LV pressure rate of decay (–dP/dt), shorter Tau, lower end-diastolic
pressure-volume relationship (EDPVR), and reduced filling pressures). Empagliflozin reduced interstitial myocardial
fibrosis at the imaging, histological and molecular level. Empagliflozin improved nitric oxide signaling (endothelial nitric
oxide synthetase [eNOS] activity, nitric oxide [NO] availability, cyclic guanosine monophosphate (cGMP) content, protein
kinase G [PKG] signaling) and enhanced titin phosphorylation (which is responsible for cardiomyocyte stiffness). Indeed,
isolated cardiomyocytes exhibited better relaxation in empagliflozin-treated animals. Myocardial consumption of glucose
and ketone bodies negatively and positively correlated with diastolic function, respectively.

CONCLUSIONS Empagliflozin ameliorates diastolic function in a nondiabetic HF porcine model, mitigates histological
and molecular remodeling, and reduces both left ventricle and cardiomyocyte stiffness.
(J Am Coll Cardiol Img 2021;14:393–407) © 2021 by the American College of Cardiology Foundation.

ISSN 1936-878X/$36.00 https://doi.org/10.1016/j.jcmg.2020.07.042

394 Santos-Gallego et al. JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021

Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

T
ABBREVIATIONS his group recently demonstrated study was to investigate the effect of empagliflozin on
AND ACRONYMS that empagliflozin ameliorates diastolic function in a nondiabetic HFrEF scenario
adverse cardiac remodeling, en- and on the pathways causing diastolic dysfunction.
CMR = cardiac magnetic
resonance
hances myocardial energetics, and improves
MATERIALS AND METHODS
EDPVR = end-diastolic
left ventricular systolic function in a nondia-
pressure-volume relationship betic porcine model (1). It was shown that
STUDY DESIGN. The study design is presented in
eNOS = endothelial nitric oxide this is mediated by a metabolic shift away
Supplemental Figure 1. Procedures were approved by
synthetase from the use of myocardial glucose toward
the Institutional Animal Care and Use Committee
HF = heart failure enhanced cardiac consumption of ketones,
(IACUC) at Icahn School of Medicine at Mount Sinai.
IMF = interstitial myocardial free fatty acids, and branched chain amino
fibrosis The proximal left anterior descending artery was
acids (BCAA) (1). However, the effects of
LV = left ventricular
occluded with a percutaneous balloon catheter for 2
empagliflozin on diastolic function have not
h, as previously reported by this group (1,20). One day
MI = myocardial infarction been yet studied.
after myocardial infarction (MI), animals were ran-
NO = nitric oxide Diastolic dysfunction is associated with
domized to empagliflozin (10 mg/day, n ¼ 7) or pla-
SGLT2 = sodium glucose adverse outcomes in both heart failure with
cebo (n ¼ 7), and treatment was maintained for 2
cotransporter type 2 preserved ejection fraction (HFpEF) (2,3) and
months. At 2 months, 3-dimensional (3D) echocardi-
T2DM = type 2 diabetes even in healthy individuals (4–7). Impor-
mellitus ography and cardiac magnetic resonance (CMR) were
tantly, in HF with reduced EF (HFrEF), dia-
Tau = time constant of completed; and cardiac catheterization was per-
stolic dysfunction also predicts adverse
ventricular pressure decay formed to evaluate cardiac function (pressure-vol-
prognosis. Recent studies demonstrate that
ume loops) and to assess myocardial metabolite
diastolic impairment predicts mortality in HFrEF
consumption (by simultaneous blood sampling from
(8,9), independent of presentation characteristics (8)
coronary arteries and coronary sinuses). Animals
and left ventricular EF (LVEF) (9); this confirms
were subsequently sacrificed, and tissue samples
classic observational studies showing the prognostic
were collected for assessment of histological and
value of diastolic function in HFrEF (10–14).
molecular remodeling. All analyses were performed
Furthermore, diastolic dysfunction also correlates
by investigators blinded to the treatment arm.
with exercise intolerance (15). Therefore, whether
SGLT2 inhibitors (SGLT2i) improved diastolic func- ASSESSMENT OF DIASTOLIC FUNCTION.
tion in HFrEF is of the highest clinical relevance. Echocardiographic evaluation of diastolic function
Two main pathophysiological mechanisms are was performed according to the American Society
responsible for diastolic dysfunction. The first mech- of Echocardiography guidelines (21). Global LV
anism is increased myocardial collagen deposition that deformation was assessed by 3D speckle tracking
causes interstitial myocardial fibrosis (IMF) and analysis using 4D LV analysis software (TomTec,
increased LV stiffness. The second mechanism in- Munich, Germany) (1,20). Strain imaging diastolic in-
volves comorbidities triggering a pro-oxidant state dex (SI-DI) was performed according to Ishii et al. (22).
that causes coronary microvascular endothelial Assessment of diastolic function using CMR was
dysfunction, which reduces nitric oxide (NO) performed by semiautomatic generation of the time-
bioavailability, cyclic monophosphate (cGMP) con- volume curve (23): peak filling rate (PFR) and first
tent, and protein kinase G (PKG) activity in adjacent filling volume (FFV), corresponding to ventricular
cardiomyocytes (16); this low PKG activity reduces suction (i.e., active relaxation of the LV).
phosphorylation of titin (the giant intracellular protein Invasive hemodynamics were additionally used to
responsible for cardiomyocyte-based stiffness), thus evaluate diastolic function. Left atrial pressure was
stiffening the cardiomyocytes (16). The effect of directly measured by a catheter after transseptal
empagliflozin on those pathways has not been studied. puncture. Pressure-volume loops were obtained by a
SGLT2i reduces HF hospitalizations (17), but the Millar Micro-Tip micromanometer catheter system
mechanism(s) of these cardiac benefits remain un- (Millar Instruments, Houston, Texas) as previously
defined (18,19). Therefore, the aim of the present reported by the present group (20,24–26).

From the Department of Cardiology, Mount Sinai School of Medicine, New York, New York, USA.
The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’
institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information,
visit the Author Center.

Manuscript received November 6, 2019; revised manuscript received July 8, 2020, accepted July 31, 2020.
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 395
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

F I G U R E 1 Empagliflozin Improves Diastolic Function Evaluated by Echocardiography

A B C
15.0 80 150.0
p = 0.001 p < 0.01 p < 0.01

Propagation Velocity Color M - Mode (cm/s)

137.5
70
12.5 125.0
60 112.5
10.0 100.0
50

IVRT (ms)
87.5
e’ (cm/s)

7.5 40 75.0
62.5
30
5.0 50.0
20 37.5
2.5 25.0
10
12.5
0.0 0 0.0
CON EMPA CON EMPA CON EMPA

D E F
1.00 15.0 50
p = 0.001 p < 0.01 p = 0.03

Left Atrial Volume 3D-Echocardiography (ml)

Tei-Index (Myocardial Performance Index)

45
12.5
40
0.75
35
10.0
30
E/e’ Ratio

0.50 7.5 25

20
5.0
15
0.25
10
2.5
5

0.00 0.0 0
CON EMPA CON EMPA CON EMPA

(A) e0 velocity. (B) Color M-mode propagation velocity. (C) Isovolumic relaxation time (IVRT). (D) Myocardial performance Tei index. (E) E/e0 ratio (surrogate of
instantaneous LV filling pressures). (F) Left atrial volume (surrogate of chronic LV filling pressures) calculated by 3-dimensional (3D) echocardiography. CON ¼ control;
EMPA ¼ empagliflozin.

ONLINE ONLY DATA SUPPLEMENT. The protocols for Differences were considered statistically significant if
MI induction, drug administration, CMR, echocardi- p < 0.05.
ography, invasive hemodynamics, cardiomyocyte
RESULTS
relaxation, histology, and molecular biology are out-
lined in the Supplemental Appendix.
ANIMAL HFrEF MODEL. Female Yorkshire pigs
STATISTICS. Results are presented as median and (n ¼ 20; weight 20  1 kg) underwent MI induction.
interquartile range. Values were compared between Six animals died during left anterior descending ar-
groups using the nonparametric Mann-Whitney U tery occlusion. The remaining animals were ran-
post hoc analysis with the Dunn-Sidak procedure domized to receive empagliflozin or placebo (n ¼ 7
performed when the overall analysis of a multiple per group) on day 1 after MI. No animals died after
group comparison was significant. Correlations were randomization.
performed using the nonparametric Spearman test. There were no differences between wither group at
All statistical calculations were performed using baseline (pre-MI). All animals exhibited HFrEF after
Prism 6.0. software (GraphPad, San Diego, California). MI but there were no differences between either
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F I G U R E 2 Empagliflozin Improves Diastolic Function Evaluated by Speckle-Tracking Strains

A B C
1.0 0.40 400
p = 0.03 p = 0.01 p = 0.02
0.35 350
0.8
0.30 300

0.25 250
0.6

E / SRE (m)
SRIVR (s−1)
SRE (s−1)

0.20 200

0.4 0.15 150

0.10 100
0.2
0.05 50

0.0 0.00 0
CON EMPA CON EMPA CON EMPA

D E F
900 p = 0.02 75.0 p = 0.02 80 p = 0.03
800
Strain Imaging Diastolic Index (SI-DI)

70
62.5
700
60

Untwisting Rate (°/s)

600 50.0
50
E / SRIVR (m)

500
37.5 40
400
30
300 25.0
20
200
12.5
100 10

0 0.0 0
CON EMPA CON EMPA CON EMPA

(A) Strain rate during early diastole (SRE). (B) Strain rate during isovolumic relaxation (SRIVR). (C) E/SRE ratio. (D) E/SRIVR ratio. (E) Strain imaging diastolic
index (SI-DI). (F) untwisting rate.

group (Supplemental Tables 2 and 3). Because the relaxation) in the treated animals (Figure 1C). More-
initial cardiac damage was similar in both groups, over, empagliflozin reduced myocardial performance
differences in diastolic function 2 months post-MI Tei index (a Doppler index combining diastolic and
could only be due to the effect of empagliflozin. systolic myocardial performance) (Figure 1D), which
EMPAGLIFLOZIN AMELIORATES DIASTOLIC DYSFUNCTION: translates to a healthier heart.
ECHOCARDIOGRAPHY. Conventional echocardiogra- Echocardiography also demonstrated lower LV
phy documented significantly improved diastolic filling pressures (surrogate of improved diastolic
function 2 months post-MI in the SGLT2i group function) at 2 months post-MI in the treated group
compared with controls. Importantly, e0 (a direct versus that in controls. E/e 0 ratio (surrogate marker of
measurement of diastolic function) (21) and color M- instantaneous LV filling pressures) (21) was lower in
mode propagation velocity were higher (i.e., better empagliflozin animals (Figure 1E). Left atrial volume,
LV relaxation) in the empagliflozin-treated group a marker of chronic elevation in left atrial pressures
versus controls (Figures 1A and 1B). Isovolumic (LAP) were evaluated by 3D echocardiography and
relaxation time, determined by echocardiography, was also smaller in the empagliflozin group versus
was also shorter (meaning more preserved LV controls (Figure 1F).
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 397
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

F I G U R E 3 Empagliflozin Improves Diastolic Function Evaluated by CMR

A B C
0.40 50 60
p = 0.02 p = 0.01 p = 0.01
45
0.35
50
40

Left Atrial Volume CMR (ml)

0.30

First Filling Volume (ml)

Peak Filling Rate (ml/s)

35
40
0.25
30

0.20 25 30

20
0.15
20
15
0.10
10
10
0.05
5

0.00 0 0
CON EMPA CON EMPA CON EMPA

D
150
PFR 0.14
Left Ventricular Volume (mL)

140 FFV 18.9

FFV/TFV 0.49
130

120

110
PFR 0.24
100 FFV 39.5
FFV/TFV 0.68
90

80
200 400 600 800 1,000
Time (ms)

(A) Peak filling rate (PFR). (B) First filling volume (during ventricular suction/active relaxation). (C) Time-volume curves of an empagliflozin-treated and a control
animal. (D) Left atrial volume. CMR ¼ cardiac magnetic resonance; FFV ¼ first filling volume; TFV ¼ total filling volume.

Speckle tracking strain analysis also confirmed demonstrated with echocardiography in the empa-
better diastolic function in empagliflozin-treated gliflozin group versus the placebo group. PFR and
pigs. Strain rate during both early diastole (SR E ) and FFV (i.e., the amount of LV filled during the active
isovolumic relaxation (SR IVR), the most important relaxation or ventricular suction) were higher in the
strain imaging indices of diastolic function, were treated animals than in controls (Figures 3A to 3C).
higher (meaning more preserved diastolic function) Left atrial volume determined by the gold standard
than in controls (Figures 2A and 2B). The E/SR E and CMR (Figure 3D) showed reduced LA volume than
E/SR IVR ratios were lower with treatment, suggesting controls, which confirms decreased chronic LAP with
lower filling pressure with SGLT2i (Figures 2E and 2F). SGLT2i.
Finally, the strain imaging diastolic index (SI-DI, ratio EMPAGLIFLOZIN AMELIORATES DIASTOLIC DYSFUNCTION:
of strain values at aortic valve closure and one-third INVASIVE HEMODYNAMICS. Pressure-volume loops
of diastole) (22) and the untwisting rate were higher observed by using micromanometer further corrobo-
with empagliflozin (Figures 2C and 2D), which in- rated the superior LV diastolic performance in the
dicates more preserved relaxation. empagliflozin versus placebo groups as demonstrated
EMPAGLIFLOZIN AMELIORATES DIASTOLIC DYSFUNCTION: by significantly enhanced peak LV pressure rate of
CMR. Evaluation of the time-volume filling curve by decay (dP/dtmax ) (Figure 4A) and shorter Tau (the time
CMR corroborated the improved diastolic function constant of ventricular pressure decay) (Figure 4B).
398 Santos-Gallego et al. JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021

Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

F I G U R E 4 Empagliflozin Improves Diastolic Function Evaluated by Invasive Hemodynamics

A B
0 100
p = 0.01 p = 0.02

90
–500
80

70
– dP/dt Min (mm Hg/s)

–1,000

Tau Weiss (ms)

60

–1,500 50

40
–2,000
30

20
–2,500
10

–3,000 0
CON EMPA CON EMPA
CON EMPA CON EMPA

C D
1.0 p = 0.03 16
p = 0.04 p = 0.02

14
0.8
12

0.6 10
EDPVR

mm Hg

0.4
6

4
0.2

0.0 0
CON EMPA Mean Left Atrial Left Ventricular End
Pressure Diastolic Pressure
CON EMPA CON EMPA

(A) Peak LV pressure rate of decay (dP/dtmin). (B) Tau (time constant of ventricular pressure decay). (C) End-diastolic pressure-volume
relationship (EDPVR). (D) Left atrial pressure (by transseptal puncture) and left ventricular end-diastolic pressure (LVEDVP).

End-diastolic pressure-volume relationship (EDPVR) EMPAGLIFLOZIN AMELIORATES INTERSTITIAL MYOCARDIAL

directly estimates LV stiffness and was lower in FIBROSIS. Empagliflozin-treated animals exhibited
treated animals (Figure 4C) than in controls, hence, smaller cardiomyocyte perimeters (measured by
empagliflozin reduces cardiac stiffness. Finally, both vinculin immunohistochemistry) (Figures 5A to 5C)
LVEDP and LAP (directly measured by transseptal than controls. This indicates less compensatory
puncture) corroborated lower filling pressure in the hypertrophy on a cellular level in the SLGT2i
treated group (Figure 4D) than in controls. group.
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 399
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

F I G U R E 5 Empagliflozin Ameliorates Interstitial Myocardial Fibrosis and Cardiomyocyte Hypertrophy

C
200.0
A B p = 0.01

187.5

Cardiomyocyte Perimeter (μm)

175.0

162.5

150.0
50µm 50µm
137.5

125.0

112.5

100.0
CON EMPA

F
20.0
p = 0.03
D E
17.5
Interstitial Myocardial Fibrosis (%)
15.0

12.5

10.0

7.5

50µm 50µm 5.0

2.5

0.0
CON EMPA

(A, B) Measurement of cardiomyocyte size. Representative images of vinculin immunohistochemistry (cardiomyocyte membrane in green; DAPI for nuclei in
blue) depicting the cardiomyocyte size of empagliflozin-treated (B) and control (A) animals. (C) Empagliflozin-treated pigs showed significantly less
cardiomyocyte hypertrophy in the remote noninfarcted myocardium than controls. (D, E) Interstitial myocardial fibrosis. Representative images of Picrosirius
Red staining in bright-field microscopy (collagen in red) of empagliflozin-treated (E) and control pig (D). (F) Empagliflozin-treated pigs showed significantly
less IMF in the remote noninfarcted myocardium than controls. (G) Quantification of extracellular volume (ECV) using T1 mapping. (H) Collagen con-
centration (using Sircol assay). (I) Hydroxyproline (a biochemical surrogate marker of collagen deposition). (J) PCR for type-1 collagen. (K) PCR for TGF-beta.
(L) Western blot for phosphorylation of SMAD2/3. IMF ¼ interstitial myocardial fibrosis; PCR ¼ polymerase chain reaction; other abbreviations as in Figure 1.

Continued on the next page

The first major pathway causing diastolic hydroxyproline (an essential amino acid found only
dysfunction is increased IMF. Picrosirius Red stain- in collagen and thus an indirect marker of IMF)
ing demonstrated a reduction in collagen deposition (Figure 5I). Molecular biology further confirmed this
in empagliflozin-treated pigs versus controls reduced IMF as the treated group exhibited reduced
(Figures 5D to 5E). This reduced histological fibrosis gene expression of the stiffer Type-1 collagen
was paralleled by smaller extracellular volume using (Figure 5J) and profibrotic cytokine TGF-b
T1 mapping (Figure 5G). The SGLT2i group showed (Figure 5K), and lower TGF-b activity (less phos-
lower levels of both biochemically determined phoSmad, 2 of 3, the downstream pathway of TGF- b)
collagen (Sircol assay) (Figure 5H) and (Figure 5L).
400 Santos-Gallego et al. JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021

Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

F I G U R E 5 Continued

G H I
40 175 80
p = 0.02 p = 0.03 p = 0.02

35 70

Hydroxyproline (mg/gr Wet Tissue)

150
Extracellular Volume (%)

30 60
125
25 50
100
20 40
75
15 30
50
10 20

5 25 10

0 0 0
CON EMPA CON EMPA CON EMPA

J K L
1.8 0.40
p < 0.01 p = 0.03 p = 0.02
0.9
Type-1 Collagen Gen Expression (PCR)

1.6 0.35
TGF-β Gene Expression (PCR)

pSMAD2/3 / Total SMAD2/3

1.4 0.8
0.30
1.2 0.7
0.25
1.0
0.20 0.6
0.8
0.15 0.5
0.6
0.10 0.4
0.4

0.2 0.05 0.3

0.0 0.00 0.2

CON EMPA CON EMPA CON EMPA

EMPAGLIFLOZIN REDUCES OXIDATIVE STRESS, concentration and PKG activity were indeed higher in
ENHANCES NO METABOLISM, AND AMELIORATES myocardium of empagliflozin-treated pigs than in
TITIN PHOSPHORYLATION. The second mechanism controls (Figures 7C and 7D).
causing diastolic dysfunction involved impairment in Reduced titin phosphorylation raised car-
the endothelial nitric oxide synthetase (eNOS)/NO/ diomyocyte stiffness and caused diastolic dysfunc-
cGMP/PKG/titin pathway, thus increasing car- tion. Importantly, empagliflozin animals exhibited
diomyocyte stiffness. Importantly, empagliflozin increased titin phosphorylation (Figure 7E) versus
improved all these steps. controls, which explains the ameliorated relaxation
Myocardial oxidative stress was reduced in the with SGLT2i. Titin is dephosphorylated by protein
myocardium of empagliflozin-treated pigs because phosphatase 1 (PP1) (27); PP1 expression was reduced
nuclear oxidative stress (assessed with 8- in empagliflozin animals versus controls (Figure 7F),
hydroxydeoxyguanosine staining) was decreased which enhances titin phosphorylation.
(Figures 6A and 6B); malondialdehyde (a marker of
lipid oxidative stress) was reduced (Figure 6C); and ASSOCIATION BETWEEN MYOCARDIAL METABOLISM
the activity of the antioxidant enzyme superoxide AND DIASTOLIC FUNCTION. Myocardial glucose con-
dismutase was increased (Figure 6D). sumption is associated with impaired diastolic
Empagliflozin increased the phosphorylation (i.e., function as shown by significant associations
activation) of eNOS (Figure 7A), leading to higher NO with the myocardial performance Tei index:
content and availability (Figure 7B). Moreover, cGMP r ¼ 0.54; SR E : r ¼ 0.61; E/SR E : r ¼ 0.71; SI-DI:
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 401
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

F I G U R E 6 Empagliflozin Mitigated Myocardial Oxidative Stress

A
DAPI 8-OHdG 8-OHdG
DAPI
EMPAGLIFLOZIN

DAPI 8-OHdG 8-OHdG

DAPI
CONTROL

B C D
30 30 0.8
p = 0.01 p = 0.02 p < 0.01
0.7
25 25
0.6
MDA / TBARS (nmol/g)

SOD Activity (U/ml)

20 20
0.5
8-OHdG

15 15 0.4

0.3
10 10
0.2
5 5
0.1

0 0 0.0
CON EMPA CON EMPA CON EMPA

(A) Representative images of immunohistochemistry for 8-hydroxydeoxyguanosine (8-OHdG in green; DAPI in blue). (B) Quantification of nuclear
oxidative stress assessed with 8-OHdG immunohistochemistry. (C) Quantification of oxidative stress measured using malondialdehyde (MDA). (D) Activity
of the antioxidant enzyme superoxide dismutase (SOD). TBARS ¼ thiobarbituric acid reactive substances; other abbreviations as in Figure 1.

r ¼ 0.57; LVEDP: r ¼ 0.71; LAP: r ¼ 0.67); E/ velocity: r ¼ 0.56; myocardial performance Tei index:
ératio: r ¼ 0.59; EDPVR: r ¼ 0.52; FFV: r ¼ 0.54; r ¼ 0.57; SRIVR: r ¼ 0.61; E/SRE : r ¼ 0.52; E/SRIVRT :
eNOS phosphorylation; r ¼ 0.46; NO levels: r ¼ 0.57; LVEDP: r ¼ 0.76; LAP: r ¼ 0.7; E/e 0 :
r ¼ 0.52; and titin phosphorylation: r ¼ 0.46; r ¼ 0.57; LA volume: r ¼ 0.53; Tau: r ¼ 0.52; LV
p < 0.05 for all (Supplemental Appendix). stiffness/EDPVR: r ¼ 0.62; PFR: r ¼ 0.71; FFV:
Myocardial ketone consumption is associated with r ¼ 0.58; eNOS: r ¼ 0.52; NO: r ¼ 0.54, and titin
better diastolic function: color M-mode propagation phosphorylation: r ¼ 0.54 (p < 0.05 for all).
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Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

F I G U R E 7 Empagliflozin Improves Nitric Oxide-Related Molecular Pathways

A B C
1.0 0.8 1.6
p = 0.02 p = 0.01 p = 0.01
0.9 0.7 1.4

NO Level (nM/mg Protein, 540 nm)

0.8
Phospho-eNOS / Total eNOS

0.6 1.2

cGMP Level (pmol/mg)

0.7
0.5 1.0
0.6

0.5 0.4 0.8

0.4 0.3 0.6

0.3
0.2 0.4
0.2
0.1 0.2
0.1

0.0 0.0 0.0

CON EMPA CON EMPA CON EMPA

D E F
6,000 80 0.8
p = 0.02 p = 0.03 p = 0.02
5,500
70 0.7
5,000
Total Titin Phosphorylation (%)

4,500 60 0.6
PKG Activity (FU/mg)

PP1 Expression (AU)

4,000
50 0.5
3,500
3,000 40 0.4
2,500
30 0.3
2,000
1,500 20 0.2
1,000
10 0.1
500
0 0 0.0
CON EMPA CON EMPA CON EMPA

(A) Phosphorylation (activation) of endothelial nitric oxide synthetase (eNOS). (B) Myocardial nitrite/nitrate ratio, a surrogate marker of nitric oxide (NO) content. (C)
Myocardial cyclic guanosine monophosphate (cGMP) level. (D) Protein kinase G (PKG) activity. (E) Phosphorylation of titin. (F) activity of protein phosphatase-1 (PP1).

Myocardial consumption of BCAA is associated of SL shortening (dSL/dtmin )” spell out SL at first

with better diastolic function: color M-Mode propa- mention and then dSL/dt min . The minimal value of
gation velocity: r ¼ 0.56; myocardial performance Tei the first derivative of SL shortening (dSL/dt min ),
index: r ¼ 0.57; SR IVR: r ¼ 0.61; E/SR E : r ¼ 0.52 which indicates the faster rate of sarcomere relaxa-
(p < 0.05); E/SR IVRT : r ¼ 0.57); LVEDP: r ¼ 0.76; tion (or re-lengthening), shows that control car-
LAP: r ¼ 0.7; E/e 0 ratio: r ¼ 0.57; LA volume: diomyocytes consistently exhibit defective relaxation
r ¼ 0.53; Tau: r ¼ 0.52; LV stiffness/EDPVR: (negative lusitropy) compared with empagliflozin
r ¼ 0.62; PFR: r ¼ 0.71; FFV: r ¼ 0.58; eNOS: r ¼ 0.52; cardiomyocytes (Figure 8C). Treatment with empa-
NO: r ¼ 0.54; and titin phosphorylation: r ¼ 0.54 gliflozin not only prevented the prolongation of
(p < 0.05 for all). tt90% but also significantly improved all phases of
sarcomere relaxation (Figures 8A to 8C).
EMPAGLIFLOZIN AMELIORATES RELAXATION OF
CARDIOMYOCYTES IN VITRO. At the cardiomyocyte DISCUSSION
level (primary culture), control animals exhibited
reduced ability to relax mainly during the latter part The present group recently reported the benefits of
of the relaxation phase (tt90%) (Figures 8A to 8C). empagliflozin on LV systolic function in a nondiabetic
Author: in “The minimal value of the first derivative HF porcine model (1). In the present study, these
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 403
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

F I G U R E 8 Empagliflozin Improves Sarcomere Relaxation in Isolated Cardiomyocytes

A B
Time (Sec) 0.45
0 0.1 0.2 0.3 0.4 0.5
0 0.4

SL Relaxation tt90% (Sec)

0.35
–20
0.3
Normalized SL (%)

–40 0.25

–60 0.2
0.15
–80
0.1
–100
0.05
–120 0
NonMI Placebo EMPA 1Hz 2Hz 3Hz
Control EMPA NonMI

C D
1Hz 2Hz 3Hz
0.5 0

* * *
0.4 * †
dSL / dtmin (µm/msec)
SL Relaxation (Sec)

† * † –1
0.3

0.2 †
–2

0.1 † †

0 –3
tt10% tt50% tt90% Control EMPA NonMI
Control EMPA NonMI

(A) Normalized sarcomere contraction and relaxation re-constructed by using average dynamic parameters at 1Hz in cardiomyocytes from non-myocardial
infarction (MI) with normal relaxation, empagliflozin (EMPA)-treated and placebo-treated animals. (B) Time to 90% of relaxation in response to pacing for
all groups. (C) Average time to different degrees of the relaxation: tt10%, tt50%, and tt90%. (D) Maximal relaxation rate (dSL/dtmin) in cardiomyocytes
isolated from every group in response to pacing. *p < 0.05 empagliflozin versus control; †p < 0.05 empagliflozin versus non-MI animals. SL ¼ sarcomere
length; tt ¼ time to.

findings were extended, and the effect of empagli- increases titin phosphorylation compared with con-
flozin on both global diastolic functions were exam- trols, which results in reduced cardiomyocyte stiff-
ined using multimodality evaluation and on the ness (Central Illustration). The combination of
specific mechanistic pathways causing diastolic reduced IMF and decreased cardiomyocytes stiffness
dysfunction. The first important finding of the pre- leads to improved diastolic function.
sent study is that empagliflozin significantly amelio- Diastolic dysfunction is an important prognostic
rated LV diastolic function versus controls in a parameter. Even in healthy patients without HF,
nondiabetic HF porcine model as demonstrated using diastolic dysfunction predicts all-cause mortality
different but complementary techniques such as (4–6) and HF (7), with worse survival according to
conventional echocardiography, strains, CMR, and the severity of diastolic dysfunction. In patients
invasive hemodynamics. The second important with HFpEF, diastolic dysfunction is associated with
finding is that empagliflozin improved both major impaired outcomes independent of comorbidities
mechanisms involved in diastolic dysfunction. On (4–6); interestingly, improvement in diastolic func-
one side, empagliflozin reduces IMF. On the other tion is associated with better survival (4). It should
side, empagliflozin activates eNOS, improves NO be highlighted that, in patients with HFrEF (a dis-
bioavailability, enhances cGMP/PKG pathways, and ease perfectly recapitulated by our model), diastolic
404 Santos-Gallego et al. JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021

Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

C E N T R A L IL L U ST R A T I O N Empagliflozin Improves Sarcomere Relaxation in Isolated

Cardiomyocytes

Empagliflozin

Porcine model of HFrEF

Urinary Glc

oxidative stress eNOS

Reduced Interstitial NO cGMP PKG
Myocardial Fibrosis Titin phosphorylation
cardiomyocyte stiffness

Echocardiography
Improved diastolic function CMR

Invasive hemodynamics
Santos-Gallego, C.G. et al. J Am Coll Cardiol Img. 2021;14(2):393–407.

Benefits of the SGLT2 inhibitor empagliflozin on diastolic function in heart failure. Empagliflozin exerts 2 effects: 1) it reduces interstitial
myocardial fibrosis; and 2) it enhances the eNOS/NO/cGMP/PKG/titin phosphorylation pathway, which decreases cardiomyocyte stiffness.
Both mechanisms result in improved diastolic function, as demonstrated using echocardiography, speckle-tracking strains, cardiac magnetic
resonance, and invasive hemodynamics. cGMP ¼ cyclic guanosine monophosphate; CMR ¼ cardiac magnetic resonance; eNOS ¼ endothelial
nitric oxide synthetase; HFrEF ¼ heart failure with reduced ejection fraction; NO ¼ nitric oxide; PKG ¼ protein kinase G.

dysfunction is also associated with worse outcomes The most important conclusion of this work is the
even after adjusting by LVEF (11,12,28) and corre- improvement of diastolic function by empagliflozin.
lates with exercise intolerance (15). In a recent study Improved relaxation with empagliflozin versus pla-
analyzing 12,141 patients with HFrEF, diastolic cebo is demonstrated by echocardiography (the uni-
impairment predicted mortality independent of all versal technique for evaluation of diastolic function),
HF characteristics (8). In another recent study, dia- using both conventional imaging (higher e0 , shorter
stolic dysfunction forecasted death and HF read- isovolumic relaxation time, higher color M-mode
mission regardless of LVEF (9). Even in classic medical propagation velocity), and strains (SRE , SR IVR, E/SR E ,
literature, large multicenter observational studies E/SRIVR, SI-DI, and untwisting). Altered LV compliance
confirmed the incremental prognostic value of Doppler changes the timing profiles of LV filling. CMR shows
measurements in patients with HFrEF, both in chronic that the time-volume curve was more preserved in
HFrEF (10–12) and in acute HFrEF states (13,14). Of the empagliflozin animals (faster PFR, higher FFV) than
utmost importance, diastolic dysfunction is not static controls, suggesting better relaxation. Invasive he-
and can improve or worsen; in fact, up to 32% of pa- modynamics confirmed the results of the imaging
tients have a change in diastolic function within 1 year techniques, as EDPVR (marker of LV stiffness) was
regardless of LVEF (6,7). This dynamic status of dia- lower and –dP/dt and Tau (markers of relaxation) were
stolic function is of crucial importance and justifies improved. Increased LV filling pressure, a conse-
treatment to improve diastolic function and amelio- quence of diastolic dysfunction, is the main cause of
rate outcomes. dyspnea; of note, empagliflozin reduced LV filling
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021 Santos-Gallego et al. 405
FEBRUARY 2021:393–407 Empagliflozin Ameliorates Diastolic Function

pressures versus controls assessed both directly availability may explain the improvement in diastolic
(LVEDP, and LAP by transseptal puncture) and with function. The fact that diastolic function is positively
imaging (E/e 0 for instantaneous filling pressures and associated with myocardial consumption of ketones
LA volume for chronic filling pressures). and BCAA and negatively associated with using
Improvement of diastolic function with empagli- myocardial glucose supports this hypothesis.
flozin in HFpEF was demonstrated by Connelly et al. Two specific mechanisms have been recently
(29) (in hypertensive rats) and Hammoudi et al. (30) postulated to explain diastolic dysfunction. The first
(in diabetic models). The first major novelty of the hypothesis is increased IMF due to excessive collagen
present study was that the benefits of SGLT2i in dia- deposition. Importantly, the current authors report
stolic function from HFpEF to HFrEF were expanded. that chronic empagliflozin treatment reduces IMF as
These findings by independent groups using different demonstrated with complementary techniques such
models confirmed the benefits of SGLT2i on diastolic as histology (Picrosirus Red), CMR (T1 mapping),
function. biochemistry (Sircol and hydroxyproline) and molec-
Another major novelty of the present study was the ular biology (polymerase chain reaction for TGF-b and
fact that empagliflozin improved diastolic function Type-1 collagen and Western blot for pSMAD2/3,
independent of diabetic status. Diabetic cardiomy- reflecting TGF- b activity), which parallels the reduc-
opathy is characterized by diastolic dysfunction. The tion in EDPVR. Furthermore, worse outcomes (mor-
fact that empagliflozin preserves diastolic function in tality and HF hospitalizations) are found in patients
diabetic models (30) may be due to improved glyce- with excessive IMF) (33), thus this antifibrotic effect
mic control. The importance of the present study lies of empagliflozin may explain the improved outcomes
in the fact that SGLT2i benefits were independent of reported in trials with SGLT2i (32). Whether the
diabetic status (nondiabetic pigs). antifibrotic effect of SGLT2i is through a primary ef-
The REFORM (Safety and Effectiveness of SGLT-2 fect (directly on fibroblasts) or a secondary mecha-
Inhibitors in Patients With Heart Failure and Dia- nism (the amelioration in LV remodeling and LV
betes; NCT02397421) trial (31) showed neutral results dilatation cause less LV wall stress, thus requiring
with dapagliflozin in HFrEF regarding LV remodeling. less IMF) has not yet be determined.
First, the REFORM trial did not report effects of dia- The second hypothesis explaining diastolic
stolic function. Second, REFORM trial recruited sub- dysfunction (16) focuses on coronary microvascular
jects with mild HF (nondilated LV, baseline LVEF of endothelial oxidative stress, which reduce nitric ox-
46%, 90% of the patients in New York Heart Associ- ide bioavailability, sGC activity, cGMP content, and
ation functional classes I to II), whereas the present PKG activity in adjacent cardiomyocytes; this in-
study investigated more severe HF (dilated LV and creases cardiomyocyte resting tension because of
baseline LVEF of 31%). Alternatively, the presence of titin hyperphosphorylation (27). Of the utmost
optimal medical treatment may reduce the efficacy of importance, empagliflozin improved all these steps.
SGLT2i. There are several general hypotheses for As compared with controls, empagliflozin reduced
explaining this empagliflozin-induced improvement oxidative stress, increased eNOS activation, boosted
in diastolic function. First, given that LV hypertrophy NO content, raised cGMP levels, activated PKG, and
and stiffness are the earliest abnormalities during enhanced phosphorylation of titin, which reduces
diastolic dysfunction (26), the present results of LV cardiomyocyte stiffness. In fact, the study demon-
hypertrophy mitigated with empagliflozin in the strated that isolated cardiomyocytes from control
current HFrEF model (1) may explain this enhanced animals exhibited lusitropic impairment consistent
lusitropy; this antihypertrophic benefit of empagli- with deficient myocardial relaxation, while car-
flozin was confirmed in animal models of HFpEF (29) diomyocytes from empagliflozin show ameliorated
and in type 2 diabetes mellitus patients (Effects of in vitro relaxation.
Empagliflozin on Cardiac Structure in Patients One of the main strengths of this study was the use
With Type 2 Diabetes [EMPA-HEART CardioLink-6; of an in vivo large-animal model with multimodality
NCT02998970] trial) (32). A second potential mecha- evaluation. The HFrEF porcine model offers unique
nism is the empagliflozin-induced improvement in translational value given its similarity to human
myocardial energetics. We have recently demon- anatomy and physiology. Furthermore, diastolic
strated that empagliflozin causes a metabolic shift function was studied using a multimodality approach
in myocardial fuel use from glucose towards the with 4 different but complementary techniques
ketones, fatty acids and BCAA, raising myocardial (echocardiography, CMR, invasive hemodynamics,
ATP content (1); as the energy-consuming part of and cardiomyocyte relaxation in vitro) and all
the cardiac cycle is diastole, this increased ATP reached the same conclusion of enhanced lusitropic
406 Santos-Gallego et al. JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021

Empagliflozin Ameliorates Diastolic Function FEBRUARY 2021:393–407

function, which confirms the benefits of empagli- improves the relaxation and decreases the stiffness
flozin on diastole. of the LV, reduces IMF, and enhances titin phos-
Our experimental observations mechanistically phorylation thus improving cardiomyocyte stiffness
support the positive cardiac benefits of SGLT2i and relaxation compared to control.
observed in patients with HFrEF (32). This improve-
FUNDING SUPPORT AND AUTHOR DISCLOSURES
ment in diastolic function may also be important in
patients with HFpEF, as increased cardiomyocyte This research was supported by an independent grant from Boeh-
diastolic stiffness is a central pathological finding in ringer Ingelheim Pharmaceuticals, which provided both drug and
HFpEF (34,35), and no drug has so far improved financial support for the study. The authors have reported that they
have no relationships relevant to the contents of this paper to
outcomes in patients with HFpEF. The present results
disclose.
in nondiabetic animals provided a novel rationale for
the treatment of HF, even in nondiabetic patients. In
fact, different clinical trials are investigating the ef- ADDRESS FOR CORRESPONDENCE: Dr. Juan J.
fect of empagliflozin independent of the diabetic Badimon, Cardiology, Mount Sinai School of Medicine,
status of the patient, both in HFrEF (EMPEROR- Box 1030, 1 Gustave L. Levy Place, New York, New York
REDUCED [EMPagliflozin outcomE tRial in Patients 10029-0310, USA. E-mail: [email protected].
With chrOnic heaRt Failure With Reduced Ejection
Fraction; NCT03057977]; and EMPA-TROPISM [Are PERSPECTIVES
the “Cardiac Benefits” of Empagliflozin Independent
of Its Hypoglycemic Activity? (ATRU-4); COMPETENCY IN MEDICAL KNOWLEDGE: In a
NCT03485222) trials (36); and in the HFpEF (EM- nondiabetic porcine model of HFrEF, the SGLT2
PEROR PRESERVED [EMPagliflozin outcomE tRial in inhibitor empagliflozin ameliorates diastolic function
Patients With chrOnic heaRt Failure With Preserved compared with control, mitigates interstitial myocar-
Ejection Fraction; NCT03057951]) trial. dial fibrosis, and increases titin phosphorylation thus
STUDY LIMITATIONS. First, diastolic dysfunction and ameliorating cardiomyocyte stiffness.
HF are frequently associated with comorbidities (hy-
pertension, hyperlipidemia, and so forth), which are TRANSLATIONAL OUTLOOK: This study demon-
not reflected in this animal model. Second, it is not strates the benefits of empagliflozin on diastolic
known how strong the effects of SGLT2i on cardiac function independently of diabetic status. Additional
remodeling would be on the background of standard studies are needed in 4 specific areas: to validate the
post-MI medical therapy. Furthermore, the present benefits effects of SGLT2 inhibitors in HFpEF; to
study did not examine the short-term effects of extend these benefits to the prevention of heart
empagliflozin on diastolic function but only chronic failure (and not only to the treatment of already
treatment. established heart failure); to define the specific
mechanism of benefit of empagliflozin on diastolic
CONCLUSIONS function; and to determine whether this is a class
benefit of all SGLT2 inhibitors.
Empagliflozin ameliorates diastolic function in a
nondiabetic HFrEF porcine model. Empagliflozin

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adverse postinfarction left ventricular remodeling betic rodent model of heart failure with preserved paper.