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JACC: HEART FAILURE
VOL. 4, NO. 4, 2016
ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION
PUBLISHED BY ELSEVIER
ISSN 2213-1779/$36.00
http://dx.doi.org/10.1016/j.jchf.2015.10.007
CLINICAL RESEARCH
Myocardial Microvascular Inflammatory
Endothelial Activation in Heart Failure
With Preserved Ejection Fraction
Constantijn Franssen, MD,a Sophia Chen, BSC,a Andreas Unger, PHD,b H. Ibrahim Korkmaz, MSC,a,c
Gilles W. De Keulenaer, MD, PHD,d Carsten Tschöpe, MD, PHD,e Adelino F. Leite-Moreira, MD, PHD,f
René Musters, PHD,a Hans W.M. Niessen, MD, PHD,c Wolfgang A. Linke, PHD,b Walter J. Paulus, MD, PHD,a
Nazha Hamdani, PHDa,b
ABSTRACT
OBJECTIVES The present study investigated whether systemic, low-grade inflammation of metabolic risk contributed
to diastolic left ventricular (LV) dysfunction and heart failure with preseved ejection fraction (HFpEF) through coronary
microvascular endothelial activation, which alters paracrine signalling to cardiomyocytes and predisposes them to
hypertrophy and high diastolic stiffness.
BACKGROUND Metabolic risk is associated with diastolic LV dysfunction and HFpEF.
METHODS We explored inflammatory endothelial activation and its effects on oxidative stress, nitric oxide (NO)
bioavailability, and cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signalling in myocardial biopsies of
HFpEF patients and validated our findings by comparing obese Zucker diabetic fatty/Spontaneously hypertensive heart
failure F1 hybrid (ZSF1)-HFpEF rats to ZSF1-Control (Ctrl) rats.
RESULTS In myocardium of HFpEF patients and ZSF1-HFpEF rats, we observed the following: 1) E-selectin and
intercellular adhesion molecule-1 expression levels were upregulated; 2) NADPH oxidase 2 expression was raised in
macrophages and endothelial cells but not in cardiomyocytes; and 3) uncoupling of endothelial nitric oxide synthase,
which was associated with reduced myocardial nitrite/nitrate concentration, cGMP content, and PKG activity.
CONCLUSIONS HFpEF is associated with coronary microvascular endothelial activation and oxidative stress.
These lead to a reduction of NO-dependent signalling from endothelial cells to cardiomyocytes, which can contribute
to the high cardiomyocyte stiffness and hypertrophy observed in HFpEF. (J Am Coll Cardiol HF 2016;4:312–24)
© 2016 by the American College of Cardiology Foundation.
From the aDepartment of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the
Netherlands; bDepartment of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany; cDepartment of Pathology
and Cardiac Surgery, VU University Medical Center, Amsterdam, the Netherlands; dLaboratory of Physiology, University of
Antwerp, Antwerp, Belgium; eDepartment of Cardiology and Pneumology, Charité-Universitätsmedizin Berlin, Berlin, Germany;
and the fDepartment of Physiology and Cardiothoracic Surgery, University of Porto, Porto, Portugal. This study was supported by a
Dutch Heart Foundation CVON (Cardiovasculair Onderzoek Nederland); ARENA (Approaching Heart Failure By Translational
Research Of RNA Mechanisms) grant and European Commission grant FP7-Health-2010; MEDIA (MEtabolic Road to DIAstolic
Heart Failure; 261409). The authors have reported that they have no relationships relevant to the contents of this paper to
disclose. Drs. Franssen and Chen contributed equally to this work.
Manuscript received July 13, 2015; revised manuscript received September 21, 2015, accepted October 1, 2015.
Franssen et al.
JACC: HEART FAILURE VOL. 4, NO. 4, 2016
APRIL 2016:312–24
M
etabolic risk is increasingly recognized as
mechanism of LV remodeling in HFpEF differs
ABBREVIATIONS
an important contributor to diastolic left
from heart failure with reduced ejection frac-
AND ACRONYMS
ventricular (LV) dysfunction and to heart
tion (HFrEF), where eccentric LV remodeling
failure with preserved ejection fraction (HFpEF).
results from cardiomyocyte cell death path-
Recent longitudinal noninvasive studies over a
ways
4-year time interval revealed close correlations
apoptosis, or necrosis (13). It also differs from
between diastolic LV stiffness and body mass index
aortic stenosis (AS), where concentric LV
(BMI) (1,2). These studies concluded that weight
remodeling is induced by excessive systolic
loss
wall stress (14). To establish the validity of
and
313
Microvascular Endothelial Inflammation in HFpEF
reduction
of
central
adiposity
could
such
as
accelerated
dysfunction
autophagy,
monophosphate
DM = diabetes mellitus
eNOS = endothelial nitric
oxide synthase
HFpEF = heart failure with
endothelial
opment of HFpEF. Similar evidence was provided
remodeling in HFpEF, the current study
HFrEF = heart failure with
by the ALLHAT trial (The Antihypertensive and
compared microvascular endothelial inflam-
reduced ejection fraction
Lipid-Lowering Treatment to Prevent Heart Attack
matory activation and its effects on myocar-
ICAM = intercellular adhesion
Trial), which enrolled patients with arterial hyperten-
dial oxidative stress, NO bioavailability, and
molecule
sion and 1 additional cardiovascular risk factor and
cGMP content in biopsies of myocardial tissue
NO = nitric oxide
observed that a high BMI at enrollment was the stron-
from
NOX2 = NADPH oxidase 2
gest predictor for development of HFpEF (3). This
Furthermore, we studied the ability of meta-
finding was consistent with the high prevalence of
bolic risk to induce HFpEF through myocardial
overweight and obesity in large trials and registries
microvascular endothelial inflammatory activation in
of HFpEF outcome registries, which almost uniformly
leptin-resistant, obese, hypertensive Zucker diabetic
reported a median BMI value in HFpEF patients in
fatty/Spontaneously hypertensive heart failure F1
excess of 30 kg/m2 .
hybrid (ZSF1) rats. These rats develop HFpEF pheno-
HFrEF,
and
AS
LV
cGMP = cyclic guanosine
prevent diastolic LV dysfunction and eventual devel-
HFpEF,
controlling
AS = aortic stenosis
patients.
preserved ejection fraction
PKG = protein kinase G
type after 20 weeks, which was evident from elevated
LV filling pressures with preserved LV systolic
SEE PAGE 325
function, increased lung weight because of pulmonary
obesity,
congestion, and increased stiffness of isolated myo-
endothelial inflammatory activation, evident from
cardial strips (15). At that time, a similar assessment of
adhesion molecule expression, appears to be the
myocardial microvascular endothelial inflammatory
earliest
(4).
activation and its effects on oxidative stress, NO
Endothelial inflammatory activation is associated
bioavailability, cGMP content, and PKG signalling was
with microalbuminuria, which was recently shown to
performed, and results were compared to those in age-
be associated with diastolic LV dysfunction (5) and to
matched, lean hypertensive ZSF1 rats with normal
predict development of HFpEF (6). When endothelial
diastolic LV function and no lung congestion.
In
primates
developing
manifestation
of
diet-induced
vascular
damage
inflammatory activation evolves to endothelial dysfunction, vasomotor responses become blunted as
METHODS
shown by a lower reactive hyperemic response (7),
which provides both diagnostic and prognostic in-
A detailed Methods section can be found in the Online
formation in HFpEF (8,9). In HFpEF, endothelial
Appendix.
dysfunction also closely relates to worsening of
HUMAN SAMPLES. Human HFrEF and HFpEF sam-
symptoms (10), functional capacity (10), and pre-
ples were procured from LV biopsies (HFrEF [n ¼ 43]
capillary pulmonary hypertension (11).
and HFpEF [n ¼ 36]). HFrEF and HFpEF patients were
This prominent involvement of metabolic risk and
hospitalized for HF and underwent transvascular LV
endothelial inflammatory activation recently led to a
endomyocardial biopsy because of suspected infil-
new paradigm for development of HFpEF (12). In
trative or inflammatory cardiomyopathy. Patients
accordance with this paradigm, metabolic comorbid-
were included if significant coronary artery disease
ities drive LV remodeling and dysfunction in HFpEF
(stenosis >50%) was ruled out by angiography and if
through coronary microvascular endothelial inflam-
histological analysis of the biopsy sample showed no
mation, which alters paracrine signalling from endo-
evidence of infiltrative or inflammatory myocardial
thelial cells to surrounding cardiomyocytes. It is
disease. Patients were classified as HFpEF if LVEF
especially the fall in nitric oxide (NO)-cyclic guanosine
was
monophosphate
(cGMP)-protein
was <97 ml/m 2, and LV end-diastolic pressure was
signalling
predisposes
kinase
G
(PKG)
>50%,
LV
end
diastolic
volume
index
to
>16 mm Hg (16). If LVEF was <45%, a patient was
develop hypertrophy and high diastolic resting
classified as HFrEF. AS patients (n ¼ 67) had severe AS
tension. Microvascular endothelial dysfunction as a
(mean aortic valve area ¼ 0.53 0.04 cm 2) without
that
cardiomyocytes
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Microvascular Endothelial Inflammation in HFpEF
concomitant coronary artery disease. Biopsy speci-
NITRATE/NITRITE CONCENTRATION. Concentrations of
mens from this group were procured from endomyo-
nitrite/nitrate were measured in tissue homogenates
cardial tissue resected from the septum (using the
from 10 ZSF1-Ctrls, 10 ZSF1-HFpEF, 16 AS, 16 HFpEF,
Morrow procedure) during aortic valve replacement.
and 16 HFrEF samples by using a colorimetric assay
The local ethics committee approved the study pro-
kit (BioVision, Milpitas, California).
tocol. Written informed consent was obtained from
IMMUNOELECTRON MICROSCOPIC QUANTIFICATION
all patients. Control human samples (n ¼ 4) were
procured
from
patients
with
OF 3-NITROTYROSINE. A
standard pre-embedding
life-threatening
immunogold electron microscopy protocol was used
arrhythmia, suspected infiltrative heart disease or
to quantify myocardial 3-nitrotyrosine in 6 ZSF1-
myocarditis, and a preserved LVEF without coronary
HFpEF and 6 ZSF1-Ctrl rat samples.
artery disease who had histology rule out myocarditis
or infiltrative pathology. Because of limited availability of human myocardial tissue, histological and
biochemical determinations could only be performed
in subgroups of patients, randomly selected by blinded investigators.
MYOCARDIAL PKA, PKC, PKG, cGMP, AND CaMKII
ACTIVITY. Kinase activities were assessed in myocar-
dial homogenates. Activity levels of 10 ZSF1-HFpEF
and 10 ZSF1-Ctrls were analyzed as described previously for protein kinase A (PKA) and PKC (17) and for
PKG and cGMP (18). Ca2þ/calmodulin-dependent
RAT SAMPLES. Obese ZSF1 rats were previously
protein kinase II (CaMKII) activity was determined
shown to develop HFpEF phenotype over a 20-week
using a CaMKII assay kit (CycLex; MBL Corp., Woburn,
lifespan (15) and are referred to as ZSF1-HFpEF
MA), using 10 ZSF1-HFpEF and ZSF1-Ctrl samples.
(n ¼ 8) in the present study. These rats are hypertensive and develop obesity and diabetes mellitus
(DM) because of leptin resistance. ZSF1-lean rats are
hypertensive but do not develop obesity or DM
and have no HFpEF phenotype (15). The ZSF1-lean rats
are referred to as ZSF1-Ctrls (n ¼ 8) in the present
study. All rats were sacrificed at 20 weeks of age.
WESTERN
BLOTTING. Expression
of
total
and
phospho-proteins was measured in homogenized
samples, as follows: expression of intercellular
adhesion molecule (ICAM)-1 was measured in 16 ZSF1Ctrls, 16 ZSF1-HFpEF, 4 AS, 7 HFrEF, and 4 HFpEF
samples; E-selectin in 6 AS, 8 HFrEF, and 8 HFpEF
samples; endothelial nitric oxide synthase (eNOS) in 8
ZSF1-Ctrls, 8 ZSF1-HFpEF, 5 AS, 7 HFrEF, and 7 HFpEF
samples; and kinases in 8 to 10 ZSF1-Ctrls and 8 to 10
ZSF1-HFpEF samples.
IMMUNOFLUORESCENCE. Myocardial
STATISTICAL ANALYSIS. Differences among groups
were
analyzed
by
1-way
followed
by
assessed by an unpaired Student t test. All analyses
were performed using Prism version 6.0 software
(GraphPad Inc., Lo Jolla, CA).
RESULTS
PATIENT
CHARACTERISTICS. More
HFpEF
than
HFrEF patients were hypertensive, and DM was more
prevalent in HFpEF than AS patients (Table 1). BMI
was
higher
in
HFpEF
than
in
AS
patients.
Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, diuretic agents, and
digoxin were more frequently used by HFpEF
patients than by HFrEF patients, compared to AS
patients. Aldosterone
receptor
antagonists were
ex-
used more by HFpEF patients than b AS patients, but
pression was measured by immunofluorescence in
were used even more frequently by HFrEF patients.
frozen sections of 10- mm-thick rat myocardium sam-
LV peak systolic pressure and LVEF were higher
ples from 12 ZSF1-HFpEF and 12 ZSF1-Ctrls.
in HFpEF patients than in HFrEF patients but
IMMUNOHISTOCHEMISTRY. For
ICAM-1
ANOVA,
Bonferroni-adjusted t test. Single comparisons were
immunohistochem-
were highest in AS patients. LV end-diastolic volume
ical staining of myeloperoxidase (MPO), CD68, and
index was lower in HFpEF patients than in HFrEF
NADPH oxidase-2 (NOX2), paraffin-embedded myo-
patients but were lowest in AS patients. LV end-
cardial samples from 8 ZSF1-Ctrls and 8 ZSF1-HFpEF
diastolic pressures were equally elevated in all pa-
were used for MPO and CD68 analysis; and 8 ZSF1-
tient groups.
Ctrls, 8 ZSF1-HFpEF, 4 HFpEF, and 4 Ctrl patient
MICROVASCULAR INFLAMMATION AND MACROPHAGE
samples were used for NOX2 analysis.
ACTIVATION IN HFpEF. Microvascular inflammation
MYOCARDIAL HYDROGEN PEROXIDE QUANTIFICATION.
and macrophage activation were assessed by analysis
Hydrogen peroxide (H 2O 2) was assessed in human
of expression of the vascular adhesion molecules
and rat myocardial tissue homogenates from 10 ZSF1-
ICAM-1 and E-selectin. Expression levels of both of
Ctrls, 10 ZSF1-HFpEF, 16 AS, 16 HFrEF, and 16 HFpEF
the markers were increased in HFpEF compared to
samples.
those in AS or HFrEF patients (Figures 1A and 1B),
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Microvascular Endothelial Inflammation in HFpEF
T A B L E 1 Baseline Characteristics and Hemodynamics of Patients
HFpEF
(n ¼ 36)
HFrEF
(n ¼ 43)
AS
(n ¼ 67)
Control
(n ¼ 4)
p Value
(HFpEF vs. HFrEF)
p Value
(HFpEF vs. AS)
63.8 2.0
60.0 2.1
65.3 1.6
51 4
0.21
0.57
% of males
56
70
47
25
0.24
0.53
% of hypertension
78
16
58
—
<0.0001
% of DM
47
30
26
—
Age, yrs
0.16
0.07
0.047
BMI, kg/m2
30.4 1.0
27.5 0.8
28.1 0.6
—
0.023
0.031
GFR, ml/min/1.73 m2
72.9 2.3
73.3 2.7
68.9 18.5
—
0.49
0.2
% of atrial fibrillation
14
33
2
—
0.067
0.028
% taking medications
ACEI/ARB
81
81
43
—
1.00
<0.0001
Beta-blocker
53
63
61
—
0.49
0.52
Diuretic agent
78
72
54
—
0.61
Aldosterone receptor antagonist
47
74
7
—
0.020
Digoxin
14
33
2
—
0.067
0.028
Statin
42
21
46
—
0.054
0.83
0.028
<0.0001
Metformin
17
14
3
—
0.76
0.049
Bronchodilators
17
19
9
—
1.00
0.34
Hemodynamics
HR, beats/min
75 2
82 4
74 2
80 16
0.073
LVPSP, mm Hg
166 6
120 3
223 4
135 15
<0.0001
LVEDP, mm Hg
25.1 1.1
22.3 1.4
22.8 1.4
13 4
LVEDVI, ml/m2
80 3
127 5
55 2
78 23
<0.0001
<0.0001
58.4 2.1
29.4 1.5
64.0 1.2
73 3
<0.0001
0.016
% of LVEF
0.71
0.12
<0.0001
0.21
Values are mean SD or%.
ACEI ¼ angiotensin-converting enzyme inhibitor; ARB ¼ angiotensin II receptor blocker; AS ¼ aortic stenosis; BMI ¼ body mass index; DM ¼ diabetes mellitus; HFpEF ¼ heart
failure with preserved ejection fraction; HFrEF indicates heart failure with reduced ejection fraction; HR ¼ heart rate; LVEDP ¼ left ventricular end-diastolic pressure; LVEDVI ¼
left ventricular end-diastolic volume index; LVEF ¼ left ventricular ejection fraction; LVPSP ¼ left ventricular peak-systolic pressure.
consistent
with
the
comorbidity-induced
proin-
flammatory status of HFpEF patients.
in cardiomyocytes, however, was comparable those in
both HFpEF and control patients (Figure 2C). Findings
Similarly, immunofluorescence showed increased
were confirmed in ZSF1-HFpEF myocardium by the
endothelial ICAM-1 and E-selectin expression levels
presence of more NOX2-expressing macrophages than
in ZSF1-HFpEF rats compared to those in ZSF1-
in ZSF1-Ctrl. Similar to the human findings, NOX2
Ctrls (Figures 1C and 1D). ZSF1-HFpEF rats also
expression levels were equal to those in car-
had higher myocardial CD68 and MPO expres-
diomyocytes
sion levels than ZSF1-Ctrls, indicating monocyte/
(Figure 2D). Furthermore, NOX2 expression was also
macrophage recruitment and neutrophil activation
manifest in microvascular endothelial cells of ZSF1-
(Figures 1E and 1F).
HFpEF rats (Figure 2D), indicative of a systemic
INCREASED OXIDATIVE STRESS IN HFpEF. To quan-
proinflammatory status.
of
ZSF1-HFpEF
and
ZSF1-Ctrl
rats
tify myocardial oxidative stress, H 2O 2 concentrations
DECREASED NO BIOAVAILABILITY IN HFpEF. Be-
were measured and shown to be higher in HFpEF
cause of the high oxidative stress, bioavailability of NO
patients than in HFrEF and AS patients (Figure 2A),
becomes jeopardized. NO bioavailability was therefore
again
comorbidity-induced
assessed by measurement of myocardial nitrite/nitrate
pro-inflammatory status of HFpEF patients. Findings
concentrations in human biopsy samples. Nitrite/
were reproduced in ZSF1-HFpEF rats, which also
nitrate concentration was indeed lower in HFpEF than
consistent
with
the
had increased myocardial H 2O 2 levels compared to
in AS and HFrEF samples (Figure 3A). These findings
those in ZSF1-Ctrls (Figure 2B).
were also confirmed in the rat model (Figure 3B).
To account for myocardial oxidative stress, we
Furthermore, immunogold-labeled electron mi-
compared NOX2 expression levels in HFpEF patients
croscopy allowed for quantification of 3-nitrotyrosine
with those in control subjects. Human HFpEF
in
myocardium contained more NOX2-expressing mac-
(Figure 3C). 3-Nitrotyrosine formation reflects con-
rophages than control patients (Figure 2C). Expression
centration of peroxynitrite, which is generated from
different
myocardial
cellular
compartments
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F I G U R E 1 Microvascular Inflammation in HFpEF Patients and ZSF1-HFpEF Rats
(A) ICAM-1 expression was higher in HFpEF than in AS (#p < 0.05 vs. AS;*p < 0.05 vs. HFrEF). (B) E-selectin levels were higher in HFpEF
patients than in those with HFrEF and AS (#p < 0.05 vs. AS; *p < 0.05 vs. HFrEF). Expression levels of ICAM-1 (C) and E-selectin (D) were
higher in ZSF1-HFpEF myocardium than in that of ZSF1-Ctrls (*p < 0.05). (E and F) ZSF1-HFpEF rats had higher levels of myocardial CD68 and
MPO than ZSF1-Ctrls (*p < 0.05). AS ¼ aortic stenosis; Ctrls ¼ controls; HFpEF ¼ heart failure with preserved ejection fraction; HFrEF ¼ heart
failure with reduced ejection fraction; ICAM ¼ intercellular adhesion molecule; MPO ¼ myeloperoxidase.
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Microvascular Endothelial Inflammation in HFpEF
F I G U R E 2 Oxidative Stress in HFpEF Patients and ZSF1-HFpEF Rats
(A) HFpEF patients had higher myocardial concentrations of H2O2 than AS and HFrEF (#p < 0.05 vs. AS; *p < 0.05 vs. HFrEF). (B) Myocardial
H2O2 concentrations in ZSF1-HFpEF were higher than in ZSF1-Ctrl rats (*p < 0.05). (C) Expression of NOX2 was comparable in cardiomyocytes
but tended to be higher in macrophages of HFpEF patients. (D) Expression of NOX2 was comparable in cardiomyocytes of ZSF1-Ctrl and ZSF1HFpEF rats. More NOX2-expressing macrophages were observed in ZSF1-HFpEF rats (*p < 0.05 vs. controls). NOX2 ¼ NADPH oxidase 2; other
abbreviations as in Figure 1.
superoxide anion and NO. 3-Nitrotyrosine expression
patients (Figure 4A). In ZSF1-HFpEF rats, there was a
was
car-
similar trend toward higher expression of the eNOS
diomyocytes (Figures 3D and 3E). Its endothelial
monomer than in ZSF1-Ctrl rats (Figure 4B). Levels of
expression
ZSF1-HFpEF
eNOS dimers were equal among human groups and
(Figure 3D), probably as a result of reduced NO
between the 2 groups of rats (Figures 4C and 4D).
bioavailability, whereas cardiomyocyte expression
Phosphorylated, and hence activated, eNOS mono-
remained unaltered (Figure 3E).
mers tended to be higher in HFpEF than in HFrEF and
UNCOUPLING OF NITRIC OXIDE SYNTHASE IN HFpEF. Endo-
AS patients (p ¼ 0.06) and in ZSF1-HFpEF than in ZSF1-
thelial nitric oxide synthase (eNOS) produces NO as a
Ctrl rats (Figures 4E and 4F). Concentrations of active,
dimer, but “uncouples” into monomers in the presence
phosphorylated NO-producing dimer were lower in
higher
in
endothelial
tended
to
cells
decrease
than
in
in
of inflammation/oxidative stress, producing superox-
HFpEF patients than in AS (p ¼ 0.06) and HFrEF
ide anion. HFpEF patients had significantly higher
(p ¼ 0.07) patients (Figure 4G). In rats, phosphorylation
expression of the eNOS monomer than HFrEF or AS
of the eNOS dimer was similar (Figure 4H).
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F I G U R E 3 Decreased Myocardial NO-Bioavailibility in HFpEF
(A) Myocardial nitrite/nitrate concentrations were lower in HFpEF than in AS and HFrEF patients and lower in HFrEF than in AS patients (*p < 0.05
HFpEF vs. AS; #p < 0.05, HFpEF vs. HFrEF; *p < 0.05, HFrEF vs. AS). (B) Myocardial nitrite/nitrate concentrations were lower in ZSF1-HFpEF
than in ZSF1-Ctrl rats (*p < 0.05 vs. controls). (C) Immunogold-labeled electron microscopy showed myocardial localization of 3-nitrotyrosine.
(D) 3-Nitrotyrosine expression tended to decrease more in endothelial cells of ZSF1-HFpEF than those of ZSF1-Ctrl rats. (E) 3-Nitrotyrosine
expression was similar in cardiomyocytes of ZSF1-HFpEF and ZSF1-Ctrl rats. NO ¼ nitric oxide; other abbreviations as in Figure 1.
DECREASED
ACTIVITY
cGMP
IN
bioavailability,
CONCENTRATION
HFpEF. Because
soluble
PKG
cGMP, which regulates PKG activity. PKG activity and
of decreased NO
cGMP concentration were indeed lower in myocar-
guanylate
AND
cyclase
(sGC)
dium of ZSF1-HFpEF rats than in ZSF1-Ctrl rats
activity falls. This leads to reduced production of
(Figures 5A and 5B). PKG lowers cardiomyocyte
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F I G U R E 4 Endothelial eNOS in HFpEF
(A) Expression of eNOS monomer was higher in HFpEF than in AS and HFrEF and higher in HFrEF than in AS (#p < 0.05 vs. AS; *p < 0.05 vs.
HFrEF). (B) A similar trend was observed in a comparison between ZSF1-HFpEF and ZSF1-Ctrl rats. (C and D) eNOS dimer concentrations were
comparable in AS, HFrEF, and HFpEF patients as well as in ZSF1-HFpEF and ZSF1-Ctrl rats. (E and F) eNOS momomer phosphorylation tended to
be higher in HFpEF patients and in ZSF1-HFpEF rats. (G and H) eNOS dimer phosphorylation tended to be lower in HFpEF patients but was
comparable in rats. eNOS ¼ endothelial nitric oxide synthase; other abbreviations as in Figure 1.
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Microvascular Endothelial Inflammation in HFpEF
stiffness through phosphorylation of titin, the giant
higher in HFpEF patients than in AS patients. The
intracellular protein responsible for cardiomyocyte-
high prevalence of a metabolic risk profile in HFpEF
based stiffness (15,17–19). Activity and expression of
patients in the present study was consistent with
other protein kinases and phosphatases, reported to
findings of the recent MEDIA (The MEtabolic Road to
modulate titin phosphorylation (19), were measured as
DIAstolic Heart Failure) European HFpEF registry
well. Activity levels of PKC and PKA in ZSF1-HFpEF rats
(23). This registry was the first to report the preva-
were not significantly different from those in ZSF1-
lence of metabolic syndrome in HFpEF and observed
Ctrl rats (Figures 5C and 5D). There were no significant
that 85% of HFpEF patients satisfied National
differences between the expression levels of the
Cholesterol Education Program III criteria of meta-
active, phosphorylated state of extracellular signal-
bolic syndrome.
regulated kinase (ERK) and those of ZSF1-HFpEF rats
Expression of adhesion molecules favors myocar-
and Ctrl rats (Figure 5E). CaMKII also lowers car-
dial infiltration of inflammatory cells, which was
diomyocyte stiffness through titin phosphorylation
evident in the current study in HFpEF patients and
(20). CaMKII activity was increased in ZSF1-HFpEF rats
in ZSF1-HFpEF rats, respectively, by the presence of
compared to that in ZSF1-Ctrl rats (Figure 5F), and
NOX2-producing macrophages and by the high
altered CaMKII activity, therefore, fails to explain the
expression levels of of CD68 and MPO. In contrast to
increased cardiomyocyte stiffness observed in previ-
viral myocarditis, the myocardial presence of macro-
ous experiments in ZSF1-HFpEF rats (15). Finally, titin
phages in HFpEF is not accompanied by evidence
can also be affected by dephosphorylating protein
of cardiomyocyte cell death (24,25). A recent study
phosphatases (PP) such as PP1 and PP2a (19). Both of
provided an explanation for this intriguing finding,
the proteins were increased in ZSF1-HFpEF compared
observing that macrophages activated by obesity
to those in ZSF1-Ctrl rats (Figures 5G and 5H).
had a distinct proinflammatory phenotype (26).
Hitherto, macrophage activation was conceived to
DISCUSSION
proceed either to an M1 phenotype with potent pro-
The present study provides comprehensive evidence
for
microvascular
endothelial
activation,
high
oxidative stress, eNOS uncoupling, and low NO
levels in LV myocardium of HFpEF patients. These
findings were reproduced in leptin-resistant, obese,
hypertensive ZSF1 rats, which develop HFpEF after
20 weeks, in contrast to lean hypertensive ZSF1 rats,
which maintain normal LV function after a similar
time period. The present study also demonstrated
that low myocardial NO level was associated with
reduced myocardial cGMP/PKG signalling in ZSF1HFPEF rats. A similar reduction was previously
demonstrated in LV myocardium of HFpEF patients
and
shown
to
be
associated
with
titin
hypo-
phosphorylation which contributes to high myocardial diastolic stiffness (18).
MICROVASCULAR INFLAMMATION. In the present
inflammatory properties or to an M2 phenotype
with anti-inflammatory properties. However, when
macrophages are activated by obesity, a distinct
phenotype is induced with low production of proinflammatory
cytokines
because
of
peroxisome
proliferator-activated receptor g (PPAR g) partially
inhibiting induction of nuclear factor kappa-light
chain-enhancer of activated B cells (NF kB). This last
effect results from the abundance in obesity of free
fatty acids such as palmitate, which stimulate PPAR g
activity. A recent study also demonstrated that
development of HFpEF was associated with monocytosis and monocyte differentiation into M2 macrophages (27).
OXIDATIVE STRESS. Myocardial H 2 O 2 concentration
was significantly higher in HFpEF patients than
in both HFrEF and AS patients. Similarly, ZSF1-
study, myocardial expression levels of E-selectin and
HFpEF rats had higher myocardial H 2O 2 concentra-
ICAM-1 were upregulated in HFpEF patients and
tions than ZSF1-Ctrl animals. H 2O 2 results from
ZSF1-HFpEF rats (Figure 6). Upregulated myocardial
conversion of superoxide anion by superoxide dis-
expression of vascular cell adhesion molecule (VCAM)
mutase, and the high H 2O 2 concentrations therefore
and myocardial microvascular rarefaction compatible
suggested increased superoxide anion production
with microvascular inflammation was previously
in HFpEF. Possible sources of superoxide anion
reported in HFpEF (21,22). Because findings in HFpEF
production are NADPH oxidases (NOX2, NOX4),
patients were similar to those in ZSF1-HFpEF rats, we
uncoupled NO synthases (eNOS, iNOS), xanthine
attribute the endothelial inflammatory activation in
oxidase,
HFpEF patients to their metabolic risk profile. HFpEF
localization of NOX2 expression was immunohis-
patients had significantly higher BMI than AS and
tochemically visualized in myocardial tissue from
HFrEF patients, and the prevalence of DM was also
HFpEF patients and ZSF1-HFpEF rats. Upregulation
and
mitochondria
(Figure
6).
Cellular
Franssen et al.
JACC: HEART FAILURE VOL. 4, NO. 4, 2016
APRIL 2016:312–24
Microvascular Endothelial Inflammation in HFpEF
F I G U R E 5 Decreased PKG Activity and cGMP Concentration in HFpEF
(A and B) Myocardial PKG activity and cGMP concentration were lower in ZSF1-HFpEF than in ZSF1-Ctrl rats. (C and D) Comparable activity levels of
PKC and PKA were seen in ZSF1-HFpEF and ZSF1-Ctrl rats. (E) Comparable expression levels of phosphorylated ERK were seen in ZSF1-HFpEF and
ZSF1-Ctrl rats. (F) Expression of CaMKII was higher in ZSF1-HFpEF than in ZSF1-Ctrl rats. (G and H) Higher expression of PP1 and PP2a was seen in
ZSF1-HFpEF than in ZSF1-Ctrl rats (*p < 0.05). CaMKII ¼ Ca2þ/calmodulin-dependent protein kinase II; cGMP ¼ cyclic guanosine monophosphate;
ERK ¼ extracellular signal-regulated kinase; PKA ¼ protein kinase A; PKC ¼ protein kinase C; other abbreviations as in Figure 1.
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Microvascular Endothelial Inflammation in HFpEF
F I G U R E 6 Downstream Effects of Microvascular Inflammation in HFpEF Myocardium
Systemic inflammation induces inflammatory activation of the endothelium of myocardial microcirculation. This leads to enhanced endothelial
expression of adhesion molecules such as ICAM-1, VCAM, and E-selectin. Because of inflammatory activation, NOX2 is upregulated in endothelial cells. This results in oxidative stress, increased levels of H2O2, uncoupling of eNOS, decreased NO bioavailability, and formation of
3-nitrotyrosine. In cardiomyocytes, decreased NO bioavailability leads to less stimulation of sGC, reduced formation of cGMP, and diminished
PKG activity. Lack of PKG activity is associated with decreased titin phosphorylation and increased passive stiffness of cardiomyocytes. cGMP
can also be generated by NPs which activate pGC. Because of the low cGMP concentration, the latter pathway failed to compensate for the
decreased NO-bioavailability. cGMP ¼ cyclic guanosine monophosphate; eNOS ¼ endothelial nitric oxide synthase; H2O2 ¼ hydrogen peroxide;
ICAM-1 ¼ intercellular adhesion molecule-1; NO ¼ nitric oxide; NOX2 ¼ NADPH nicotinamide adenine dinucleotide phosphate oxidase 2; NPs ¼
natriuretic peptides; pGC ¼ particulate guanylate cyclase; PKG ¼ protein kinase G; sGC ¼ soluble guanylate cyclase; VCAM ¼ vascular cell
adhesion molecule.
of
macro-
reduced 3-nitrotyrosine expression in endothelial
phages and microvascular endothelium but not in
NOX2
expression
cells. 3-Nitrotyrosine formation reflects peroxynitrite
cardiomyocytes. The current findings of upregulated
concentration, which is generated from superoxide
endothelial NOX2 expression and unaltered NOX2
anion and NO. Because of increased H2O 2 concentra-
expression in cardiomyocytes in HFpEF myocar-
tion, the trend for reduced 3-nitrotyrosine in endo-
dium support the assertion that myocardial re-
thelial cells of HFpEF myocardium probably resulted
modeling
endothelial
from low NO production. The latter was consistent
activation, in contrast to HFrEF, where myocardial
with the low nitrite/nitrate concentrations observed
remodeling is driven by cardiomyocyte cell death
in
triggered
ZSF1-HFpEF rats. Reduced NO production can be
in
by
HFpEF
was
is
observed
driven
upregulated
NOX2
by
in
activity
within
cardiomyocytes (28).
myocardium
of
both
HFpEF
patients
and
explained by eNOS uncoupling, which was confirmed
NO-cGMP-PKG SIGNALLING. Similar to NOX2 ex-
in both HFpEF patients and ZSF1-HFpEF rats. eNOS
pression,
revealed
uncoupling switches eNOS from the NO producing
myocardial 3-nitrotyrosine expression in ZSF1-Ctrl
dimer to the superoxide anion generating monomer
and
in
(29). Apart from eNOS uncoupling, the present study
endothelial cells, with less expression in cardio-
also observed modified eNOS phosphorylation in
myocytes. Development of HFpEF failed to affect
HFpEF patients, with a higher phosphorylation of
3-nitrotyrosine expression in cardiomyocytes but
monomeric eNOS increasing superoxide production
immunoelectron
ZSF1-HFpEF
rats
was
microscopy
localized
mainly
Franssen et al.
JACC: HEART FAILURE VOL. 4, NO. 4, 2016
APRIL 2016:312–24
Microvascular Endothelial Inflammation in HFpEF
and a lower phosphorylation of dimeric eNOS
CONCLUSIONS
decreasing NO production. Low NO production
affects sGC activity and results in decreased levels of
Microvascular inflammatory endothelial activation,
cGMP and low PKG activity (Figure 6). This was pre-
high oxidative stress, eNOS uncoupling, and impaired
viously observed in human HFpEF myocardium
cGMP-PKG signalling were observed in LV myocar-
(18) and was currently confirmed in ZSF1-HFpEF
dium of HFpEF patients. Similar changes were
myocardium. Low PKG activity increases diastolic
reproduced in obese, leptin-resistant, hypertensive
stiffness through reduced phosphorylation of the
ZSF1 rats, which developed a HFpEF phenotype after
giant cytoskeletal protein titin (Figure 6) (30,31).
20 weeks, but not in lean, hypertensive ZSF1 rats.
The phosphorylation and distensibility of titin are
Because of these findings, myocardial microvascular
also affected by phosphatases and other kinases, such
inflammation induced by metabolic comorbidities
as PKCa , PKA, ERK and CaMKII (20,30,31). In the
could be an important contributor to development of
present study, ZSF1-HFpEF rats showed higher
HFpEF.
expression levels of PP1 and PP2a. PKC, PKA, and ERK
activity levels were comparable, but CaMKII activity
REPRINT REQUESTS AND CORRESPONDENCE: Prof.
was increased. Phosphorylation of titin by CaMKII
Dr. Walter J. Paulus, ICaR-VU, VU University Medical
augments titin distensibility (32) and the higher
Center, Van der Boechorststraat 7, 1081 BT Amster-
CaMKII activity therefore cannot explain the high
dam, the Netherlands. E-mail: [email protected].
cardiomyocyte resting tension previously observed
in ZSF1-HFpEF rats (15). The latter more likely
results from imbalanced activities of PKG and
PERSPECTIVES
phosphatases.
STUDY LIMITATIONS. Except for immunohistochem-
ical assessment of NOX2 expression, the present
study compared myocardial tissue of HFpEF patients
to tissue of HFrEF and AS patients because of limited
availability of myocardial tissue from control subjects. This limitation was partially accounted for by
inclusion of an animal model consisting of obese and
lean ZSF1 rats (15). Both HFpEF and HFrEF patients
were investigated following admission for acute heart
failure. An inflammatory component related to the
acute heart failure episode could have contributed to
COMPETENCY IN MEDICAL KNOWLEDGE: Metabolic
comorbidities such as obesity and DM induce chronic endothelial
inflammation of the coronary microvasculature, which
reduces myocardial NO bioavailability, PKG activity and cGMP
concentration. The latter predisposes to cardiomyocyte
stiffness and hypertrophy, both of which are characteristic for
HFpEF.
TRANSLATIONAL OUTLOOK: Future therapeutic strategies
in HFPEF should target the metabolic risk-induced chronic
inflammation of the myocardial microvasculature.
the microvascular inflammation.
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KEY WORDS endothelium, heart failure,
inflammation, nitric oxide, oxidative stress
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A PPE NDI X For a detailed Methods section,
please see the online version of this article.