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
initial cardiac damage was similar in both groups,
differences in diastolic function 2 months post-MI
could only be due to the effect of empagliflozin.
EMPAGLIFLOZIN AMELIORATES DIASTOLIC DYSFUNCTION:
ECHOCARDIOGRAPHY. Conventional
phy documented significantly improved diastolic
function 2 months post-MI in the SGLT2i group
compared with controls. Importantly, e0 (a direct
measurement of diastolic function) (21) and color M-
mode propagation velocity were higher (i.e., better
LV relaxation) in the empagliflozin-treated group
versus controls (Figures 1A and 1B). Isovolumic
relaxation time, determined by echocardiography,
was also shorter (meaning more preserved LV
relaxation) in the treated animals (Figure 1C). More-
over, empagliflozin reduced myocardial performance
Tei index (a Doppler index combining diastolic and
systolic myocardial performance) (Figure 1D), which
translates to a healthier heart.
echocardiogra- Echocardiography also demonstrated lower LV
filling pressures (surrogate of improved diastolic
function) at 2 months post-MI in the treated group
versus that in controls. E/e 0 ratio (surrogate marker of
instantaneous LV filling pressures) (21) was lower in
empagliflozin animals (Figure 1E). Left atrial volume,
a marker of chronic elevation in left atrial pressures
(LAP) were evaluated by 3D echocardiography and
was also smaller in the empagliflozin group versus
controls (Figure 1F).
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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/
ératio: r ¼ 0.59; EDPVR: r ¼ 0.52; FFV: r ¼ 0.54;
eNOS phosphorylation; r ¼ 0.46;
r ¼ 0.52; and titin phosphorylation: r ¼ 0.46;
p < 0.05 for all (Supplemental Appendix).
Myocardial ketone consumption is associated with
better diastolic function: color M-mode propagation
velocity: r ¼ 0.56; myocardial performance Tei index:
r ¼ 0.57; SRIVR: r ¼ 0.61; E/SRE : r ¼ 0.52; E/SRIVRT :
NO levels: r ¼ 0.57; LVEDP: r ¼ 0.76; LAP: r ¼ 0.7; E/e 0 :
r ¼ 0.57; LA volume: r ¼ 0.53; Tau: r ¼ 0.52; LV
stiffness/EDPVR: r ¼ 0.62; PFR: r ¼ 0.71; FFV:
r ¼ 0.58; eNOS: r ¼ 0.52; NO: r ¼ 0.54, and titin
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-
gation velocity: r ¼ 0.56; myocardial performance Tei
index: r ¼ 0.57; SR IVR: r ¼ 0.61; E/SR E : r ¼ 0.52
(p < 0.05); E/SR IVRT : r ¼ 0.57); LVEDP: r ¼ 0.76;
LAP: r ¼ 0.7; E/e 0 ratio: r ¼ 0.57; LA volume:
r ¼ 0.53; Tau: r ¼ 0.52; LV stiffness/EDPVR:
r ¼ 0.62; PFR: r ¼ 0.71; FFV: r ¼ 0.58; eNOS: r ¼ 0.52;
NO: r ¼ 0.54; and titin phosphorylation: r ¼ 0.54
(p < 0.05 for all).
EMPAGLIFLOZIN AMELIORATES RELAXATION OF
CARDIOMYOCYTES IN VITRO. At the cardiomyocyte
level (primary culture), control animals exhibited
reduced ability to relax mainly during the latter part
of the relaxation phase (tt90%) (Figures 8A to 8C).
Author: in “The minimal value of the first derivative
mention and then dSL/dt min . The minimal value of
the first derivative of SL shortening (dSL/dt min ),
which indicates the faster rate of sarcomere relaxa-
tion (or re-lengthening), shows that control car-
diomyocytes consistently exhibit defective relaxation
(negative lusitropy) compared with empagliflozin
cardiomyocytes (Figure 8C). Treatment with empa-
gliflozin not only prevented the prolongation of
tt90% but also significantly improved all phases of
sarcomere relaxation (Figures 8A to 8C).
DISCUSSION
The present group recently reported the benefits of
empagliflozin on LV systolic function in a nondiabetic
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-
flozin on both global diastolic functions were exam-
ined using multimodality evaluation and on the
specific mechanistic
dysfunction. The first important finding of the pre-
sent study is that empagliflozin significantly amelio-
rated LV diastolic function versus controls in a
nondiabetic HF porcine model as demonstrated using
different but complementary techniques such as
conventional echocardiography, strains, CMR, and
invasive hemodynamics.
finding is that empagliflozin improved both major
mechanisms involved in diastolic dysfunction. On
one side, empagliflozin reduces IMF. On the other
side, empagliflozin activates eNOS, improves NO
bioavailability, enhances cGMP/PKG pathways, and
increases titin phosphorylation compared with con-
trols, which results in reduced cardiomyocyte stiff-
ness (Central Illustration). The combination of
pathways causing diastolic reduced IMF and decreased cardiomyocytes stiffness
leads to improved diastolic function.
Diastolic dysfunction is an important prognostic
parameter. Even in healthy patients without HF,
diastolic dysfunction predicts all-cause mortality
(4–6) and HF (7), with worse survival according to
the severity of diastolic dysfunction. In patients
The second important with HFpEF, diastolic dysfunction is associated with
impaired outcomes independent of comorbidities
(4–6); interestingly, improvement in diastolic func-
tion is associated with better survival (4). It should
be highlighted that, in patients with HFrEF (a dis-
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
even after adjusting by LVEF (11,12,28) and corre-
lates with exercise intolerance (15). In a recent study
analyzing 12,141 patients with HFrEF,
impairment predicted mortality independent of all
HF characteristics (8). In another recent study, dia-
stolic dysfunction forecasted death and HF read-
mission regardless of LVEF (9). Even in classic medical
literature, large multicenter observational studies
confirmed the incremental prognostic value of Doppler
measurements in patients with HFrEF, both in chronic
HFrEF (10–12) and in acute HFrEF states (13,14). Of the
utmost importance, diastolic dysfunction is not static
and can improve or worsen; in fact, up to 32% of pa-
tients have a change in diastolic function within 1 year
regardless of LVEF (6,7). This dynamic status of dia-
stolic function is of crucial importance and justifies
treatment to improve diastolic function and amelio-
rate outcomes.
The most important conclusion of this work is the
improvement of diastolic function by empagliflozin.
Improved relaxation with empagliflozin versus pla-
diastolic cebo is demonstrated by echocardiography (the uni-
versal technique for evaluation of diastolic function),
using both conventional imaging (higher e0 , shorter
isovolumic relaxation time, higher color M-mode
propagation velocity), and strains (SRE , SR IVR, E/SR E ,
E/SRIVR, SI-DI, and untwisting). Altered LV compliance
changes the timing profiles of LV filling. CMR shows
that the time-volume curve was more preserved in
empagliflozin animals (faster PFR, higher FFV) than
controls, suggesting better relaxation. Invasive he-
modynamics confirmed the results of the imaging
techniques, as EDPVR (marker of LV stiffness) was
lower and –dP/dt and Tau (markers of relaxation) were
improved. Increased LV filling pressure, a conse-
quence of diastolic dysfunction, is the main cause of
dyspnea; of note, empagliflozin reduced LV filling
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021
FEBRUARY 2021:393–407
pressures versus controls assessed both directly
(LVEDP, and LAP by transseptal puncture) and with
imaging (E/e 0 for instantaneous filling pressures and
LA volume for chronic filling pressures).
Improvement of diastolic function with empagli-
flozin in HFpEF was demonstrated by Connelly et al.
(29) (in hypertensive rats) and Hammoudi et al. (30)
(in diabetic models). The first major novelty of the
present study was that the benefits of SGLT2i in dia-
stolic function from HFpEF to HFrEF were expanded.
These findings by independent groups using different
models confirmed the benefits of SGLT2i on diastolic
function.
Another major novelty of the present study was the
fact that empagliflozin improved diastolic function
independent of diabetic status. Diabetic cardiomy-
opathy is characterized by diastolic dysfunction. The
fact that empagliflozin preserves diastolic function in
diabetic models (30) may be due to improved glyce-
mic control. The importance of the present study lies
in the fact that SGLT2i benefits were independent of
diabetic status (nondiabetic pigs).
The REFORM (Safety and Effectiveness of SGLT-2
Inhibitors in Patients With Heart Failure and Dia-
betes; NCT02397421) trial (31) showed neutral results
with dapagliflozin in HFrEF regarding LV remodeling.
First, the REFORM trial did not report effects of dia-
stolic function. Second, REFORM trial recruited sub-
jects with mild HF (nondilated LV, baseline LVEF of
46%, 90% of the patients in New York Heart Associ-
ation functional classes I to II), whereas the present
study investigated more severe HF (dilated LV and
baseline LVEF of 31%). Alternatively, the presence of
optimal medical treatment may reduce the efficacy of
SGLT2i. There are several general hypotheses for
explaining this empagliflozin-induced improvement
in diastolic function. First, given that LV hypertrophy
and stiffness are the earliest abnormalities during
diastolic dysfunction (26), the present results of LV
hypertrophy mitigated with empagliflozin in the
current HFrEF model (1) may explain this enhanced
lusitropy; this antihypertrophic benefit of empagli-
flozin was confirmed in animal models of HFpEF (29)
and in type 2 diabetes mellitus patients (Effects of
Empagliflozin on Cardiac Structure in Patients
With Type 2 Diabetes [EMPA-HEART CardioLink-6;
NCT02998970] trial) (32). A second potential mecha-
nism is the empagliflozin-induced improvement in
myocardial energetics. We have recently demon-
strated that empagliflozin causes a metabolic shift
in myocardial fuel use from glucose towards the
ketones, fatty acids and BCAA, raising myocardial
ATP content (1); as the energy-consuming part of
the cardiac cycle is diastole, this increased ATP
Santos-Gallego et al. 405
Empagliflozin Ameliorates Diastolic Function
availability may explain the improvement in diastolic
function. The fact that diastolic function is positively
associated with myocardial consumption of ketones
and BCAA and negatively associated with using
myocardial glucose supports this hypothesis.
Two specific mechanisms have been recently
postulated to explain diastolic dysfunction. The first
hypothesis is increased IMF due to excessive collagen
deposition. Importantly, the current authors report
that chronic empagliflozin treatment reduces IMF as
demonstrated with complementary techniques such
as histology (Picrosirus Red), CMR (T1 mapping),
biochemistry (Sircol and hydroxyproline) and molec-
ular biology (polymerase chain reaction for TGF-b and
Type-1 collagen and Western blot for pSMAD2/3,
reflecting TGF- b activity), which parallels the reduc-
tion in EDPVR. Furthermore, worse outcomes (mor-
tality and HF hospitalizations) are found in patients
with excessive IMF) (33), thus this antifibrotic effect
of empagliflozin may explain the improved outcomes
reported in trials with SGLT2i (32). Whether the
antifibrotic effect of SGLT2i is through a primary ef-
fect (directly on fibroblasts) or a secondary mecha-
nism (the amelioration in LV remodeling and LV
dilatation cause less LV wall stress, thus requiring
less IMF) has not yet be determined.
The second hypothesis explaining diastolic
dysfunction (16) focuses on coronary microvascular
endothelial oxidative stress, which reduce nitric ox-
ide bioavailability, sGC activity, cGMP content, and
PKG activity in adjacent cardiomyocytes; this in-
creases cardiomyocyte resting tension because of
titin hyperphosphorylation (27). Of the utmost
importance, empagliflozin improved all these steps.
As compared with controls, empagliflozin reduced
oxidative stress, increased eNOS activation, boosted
NO content, raised cGMP levels, activated PKG, and
enhanced phosphorylation of titin, which reduces
cardiomyocyte stiffness. In fact, the study demon-
strated that isolated cardiomyocytes from control
animals exhibited lusitropic impairment consistent
with deficient myocardial relaxation, while car-
diomyocytes from empagliflozin show ameliorated
in vitro relaxation.
One of the main strengths of this study was the use
of an in vivo large-animal model with multimodality
evaluation. The HFrEF porcine model offers unique
translational value given its similarity to human
anatomy and physiology. Furthermore, diastolic
function was studied using a multimodality approach
with 4 different but complementary techniques
(echocardiography, CMR, invasive hemodynamics,
and cardiomyocyte relaxation in vitro) and all
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-
flozin on diastole.
Our experimental observations mechanistically
support the positive cardiac benefits of SGLT2i
observed in patients with HFrEF (32). This improve-
ment in diastolic function may also be important in
patients with HFpEF, as increased cardiomyocyte
diastolic stiffness is a central pathological finding in
HFpEF (34,35), and no drug has so far improved
outcomes in patients with HFpEF. The present results
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-
fect of empagliflozin independent of the diabetic
status of the patient, both in HFrEF (EMPEROR-
REDUCED [EMPagliflozin outcomE tRial in Patients
With chrOnic heaRt Failure With Reduced Ejection
Fraction; NCT03057977]; and EMPA-TROPISM [Are
the “Cardiac Benefits” of Empagliflozin Independent
of Its Hypoglycemic Activity?
NCT03485222) trials (36); and in the HFpEF (EM-
PEROR PRESERVED [EMPagliflozin outcomE tRial in
Patients With chrOnic heaRt Failure With Preserved
Ejection Fraction; NCT03057951]) trial.
STUDY LIMITATIONS. First, diastolic dysfunction and
HF are frequently associated with comorbidities (hy-
pertension, hyperlipidemia, and so forth), which are
not reflected in this animal model. Second, it is not
known how strong the effects of SGLT2i on cardiac
remodeling would be on the background of standard
post-MI medical therapy. Furthermore, the present
study did not examine the short-term effects of
empagliflozin on diastolic function but only chronic
treatment.
CONCLUSIONS
Empagliflozin ameliorates diastolic function in a
nondiabetic HFrEF porcine model. Empagliflozin
improves the relaxation and decreases the stiffness
of the LV, reduces IMF, and enhances titin phos-
phorylation thus improving cardiomyocyte stiffness
and relaxation compared to control.
FUNDING SUPPORT AND AUTHOR DISCLOSURES
This research was supported by an independent grant from Boeh-
ringer Ingelheim Pharmaceuticals, which provided both drug and
financial support for the study. The authors have reported that they
have no relationships relevant to the contents of this paper to
disclose.
ADDRESS FOR CORRESPONDENCE: Dr. Juan J.
Badimon, Cardiology, Mount Sinai School of Medicine,
Box 1030, 1 Gustave L. Levy Place, New York, New York
10029-0310, USA. E-mail: juan.badimon@mssm.edu.
PERSPECTIVES
(ATRU-4); COMPETENCY IN MEDICAL KNOWLEDGE: In a
nondiabetic porcine model of HFrEF, the SGLT2
inhibitor empagliflozin ameliorates diastolic function
compared with control, mitigates interstitial myocar-
dial fibrosis, and increases titin phosphorylation thus
ameliorating cardiomyocyte stiffness.
TRANSLATIONAL OUTLOOK: This study demon-
strates the benefits of empagliflozin on diastolic
function independently of diabetic status. Additional
studies are needed in 4 specific areas: to validate the
benefits effects of SGLT2 inhibitors in HFpEF; to
extend these benefits to the prevention of heart
failure (and not only to the treatment of already
established heart failure); to define the specific
mechanism of benefit of empagliflozin on diastolic
function; and to determine whether this is a class
benefit of all SGLT2 inhibitors.

REFERENCES

1. Santos-Gallego CG, Requena-Ibanez JA, San
Antonio R, et al. Empagliflozin ameliorates
adverse left ventricular remodeling in nondia-
betic heart failure by enhancing
myocardial energetics. J Am Coll Cardiol 2019;
73:1931–44.
2. Borlaug BA, Paulus WJ. Heart failure with
preserved ejection fraction: pathophysiology,
diagnosis, and treatment. Eur Heart J 2011;32:
670–9.
3. Nagueh SF. Left ventricular diastolic function:
understanding pathophysiology, diagnosis, and
prognosis with echocardiography. J Am Coll Car-
diol Img 2020;13:228–44.
4. Achong N, Wahi S, Marwick TH. Evolution and
outcome of diastolic dysfunction. Heart 2009;95:
813–8.
5. Halley CM, Houghtaling PL, Khalil MK,
Thomas JD, Jaber WA. Mortality rate in patients
with diastolic dysfunction and normal systolic
function. Arch Intern Med 2011;171:1082–7.
6. Aljaroudi W, Alraies MC, Halley C, et al. Impact
of progression of diastolic dysfunction on mor-
tality in patients with normal ejection fraction.
Circulation 2012;125:782–8.
7. Kane GC, Karon BL, Mahoney DW, et al. Pro-
gression of left ventricular diastolic dysfunction
and risk of heart failure. JAMA 2011;306:856–63.
8. Benfari G, Miller WL, Antoine C, et al. Diastolic
determinants of excess mortality in heart failure
with reduced ejection fraction. J Am Coll Cardiol
HF 2019;7:808–17.
9. Machino-Ohtsuka T, Seo Y, Ishizu T, et al.
Clinical utility of the 2016 ASE/EACVI recommen-
dations for the evaluation of left ventricular dia-
stolic function in the stratification of post-
discharge prognosis in patients with acute heart
failure. Eur Heart J Cardiovasc Imaging 2019;20:
1129–37.
10. Pozzoli M, Traversi E, Cioffi G, Stenner R,
Sanarico M, Tavazzi L. Loading manipulations
improve the prognostic value of Doppler
JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 2, 2021
FEBRUARY 2021:393–407
evaluation of mitral flow in patients with chronic
heart failure. Circulation 1997;95:1222–30.
11. Xie GY, Berk MR, Smith MD, Gurley JC,
DeMaria AN. Prognostic value of Doppler trans-
mitral flow patterns in patients with congestive
heart failure. J Am Coll Cardiol 1994;24:132–9.
12. Somaratne JB, Whalley GA, Gamble GD,
Doughty RN. Restrictive filling pattern is a
powerful predictor of heart failure events post-
acute myocardial infarction and in established
heart failure: a literature-based meta-analysis.
J Card Fail 2007;13:346–52.
13. Pinamonti B, Zecchin M, Di Lenarda A,
Gregori D, Sinagra G, Camerini F. Persistence of
restrictive left ventricular filling pattern in dilated
cardiomyopathy: an ominous prognostic sign.
J Am Coll Cardiol 1997;29:604–12.
14. Nijland F, Kamp O, Karreman AJ, van
Eenige MJ, Visser CA. Prognostic implications of
restrictive left ventricular filling in acute myocar-
dial infarction: a serial Doppler echocardiographic
study. J Am Coll Cardiol 1997;30:1618–24.
15. Skaluba SJ, Litwin SE. Mechanisms of exercise
intolerance: insights from tissue Doppler imaging.
Circulation 2004;109:972–7.
16. Paulus WJ, Tschope C. A novel paradigm for heart
failure with preserved ejection fraction: comorbid-
ities drive myocardial dysfunction and remodeling
through coronary microvascular endothelial inflam-
mation. J Am Coll Cardiol 2013;62:263–71.
17. McMurray JJV, Solomon SD, Inzucchi SE, et al.
Dapagliflozin in patients with heart failure and
reduced ejection fraction. N Engl J Med 2019;381:
1995–2008.
18. Flores E, Santos-Gallego CG, Diaz-Mejia N,
Badimon JJ. Do the SGLT-2 inhibitors offer more
than hypoglycemic activity? Cardiovasc Drugs Ther
2018;32:213–22.
19. Garcia-Ropero A, Badimon JJ, Santos-
Gallego CG. The pharmacokinetics and pharma-
codynamics of SGLT2 inhibitors for type 2 diabetes
mellitus: the latest developments. Expert Opin
Drug Metab Toxicol 2018;14:1287–302.
20. Santos-Gallego CG, Vahl TP, Goliasch G, et al.
Sphingosine-1-phosphate receptor agonist fingo-
limod increases myocardial salvage and decreases
adverse postinfarction left ventricular remodeling
in a porcine model of ischemia/reperfusion. Cir-
culation 2016;133:954–66.
21. Nagueh SF, Smiseth OA, Appleton CP, et al.
Recommendations for the evaluation of left ven-
tricular diastolic function by echocardiography: an
update from the American Society of Echocardi-
ography and the European Association of Cardio-
vascular Imaging. J Am Soc Echocardiogr 2016;29:
277–314.
22. Ishii K, Suyama T, Imai M, et al.
Abnormal regional left ventricular systolic and
diastolic function in patients with coronary artery
disease undergoing percutaneous coronary inter-
vention: clinical significance of post-ischemic dia-
stolic stunning. J Am Coll Cardiol 2009;54:
1589–97.
23. Kawaji K, Codella NC, Prince MR, et al. Auto-
mated segmentation of routine clinical cardiac
magnetic resonance imaging for assessment of
left ventricular diastolic dysfunction. Circ Car-
diovasc Imaging 2009;2:476–84.
24. Ishikawa K, Chemaly ER, Tilemann L, et al.
Assessing left ventricular systolic dysfunction af-
ter myocardial infarction: are ejection fraction and
dP/dt (max) complementary or redundant? Am J
Physiol Heart Circ Physiol 2012;302:H1423–8.
25. Ishikawa K, Aguero J, Tilemann L, et al. Char-
acterizing preclinical models of ischemic heart
failure: differences between LAD and LCx in-
farctions. Am J Physiol Heart Circ Physiol 2014;
307:H1478–86.
26. Ishikawa K, Aguero J, Oh JG, et al. Increased
stiffness is the major early abnormality in a pig
model of severe aortic stenosis and predisposes to
congestive heart failure in the absence of systolic
dysfunction. J Am Heart Assoc 2015;4.
27. Franssen C, Chen S, Unger A, et al. Myocardial
Microvascular Inflammatory Endothelial Activation
in Heart Failure With Preserved Ejection Fraction.
J Am Coll Cardiol HF 2016;4:312–24.
28. Wang M, Yip GW, Wang AY, et al. Peak early
diastolic mitral annulus velocity by tissue Doppler
imaging adds independent and incremental prog-
nostic value. J Am Coll Cardiol 2003;41:820–6.
29. Connelly KA, Zhang Y, Visram A, et al. Empa-
gliflozin improves diastolic function in a nondia-
betic rodent model of heart failure with preserved
Santos-Gallego et al. 407
Empagliflozin Ameliorates Diastolic Function
ejection fraction. J Am Coll Cardiol Basic Trans
Science 2019;4:27–37.
30. Hammoudi N, Jeong D, Singh R, et al.
Empagliflozin improves left ventricular diastolic
dysfunction in a genetic model of type 2 diabetes.
Cardiovasc Drugs Ther 2018;31:233–46.
31. Singh JSS, Mordi IR, Vickneson K, et al. Dapa-
gliflozin versus placebo on left ventricular
remodeling in patients with diabetes and heart
failure: the REFORM trial. Diabetes Care 2020;43:
1356–9.
32. McMurray JJV, Solomon SD, Inzucchi SE, et al.
Dapagliflozin in patients with heart failure
and reduced ejection fraction. N Engl J Med
2019;381:1995–2008.
33. Wong TC, Piehler K, Meier CG, et al. Associa-
tion between extracellular matrix expansion
quantified by cardiovascular magnetic resonance
and short-term mortality. Circulation 2012;126:
1206–16.
34. Borbely A, Falcao-Pires I, van Heerebeek L,
et al. Hypophosphorylation of the stiff N2B titin
isoform raises cardiomyocyte resting tension in
failing human myocardium. Circ Res 2009;104:
780–6.
35. Westermann D, Kasner M, Steendijk P, et al.
Role of left ventricular stiffness in heart failure
with normal ejection fraction. Circulation 2008;
117:2051–60.
36. Santos-Gallego CG, Garcia-Ropero A,
Mancini D, et al. Rationale and design of the
EMPA-TROPISM trial (ATRU-4): are the “cardiac
benefits” of empagliflozin independent of its hy-
poglycemic activity? Cardiovasc Drugs Ther 2019;
33:87–95.
KEY WORDS animal models, diabetes,
diastolic function, heart failure, cardiac
remodeling, myocardial metabolism, SGLT2
inhibition
A PP END IX For supplemental figures and
tables, please see the online version of this
paper.