Cardiac Inflammation Contributes to Changes in the Extracellular Matrix in Patients

With Heart Failure and Normal Ejection Fraction

Dirk Westermann, Diana Lindner, Mario Kasner, Christine Zietsch, K. Savvatis, F. Escher,
J. von Schlippenbach, C. Skurk, Paul Steendijk, Alexander Riad, Wolfgang Poller,
Heinz-Peter Schultheiss and Carsten Tschpe
Circ Heart Fail 2011;4;44-52; originally published online November 12, 2010;
DOI: 10.1161/CIRCHEARTFAILURE.109.931451
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Cardiac Inflammation Contributes to Changes in the

Extracellular Matrix in Patients With Heart Failure and
Normal Ejection Fraction
Dirk Westermann, MD*; Diana Lindner, PhD*; Mario Kasner, MD; Christine Zietsch, PhD;
K. Savvatis, MD; F. Escher, MD; J. von Schlippenbach, MD; C. Skurk, MD; Paul Steendijk, PhD;
Alexander Riad, MD; Wolfgang Poller, MD; Heinz-Peter Schultheiss, MD; Carsten Tschope, MD
BackgroundThe pathophysiology of heart failure with normal ejection fraction (HFNEF) is still under discussion. Here
we report the influence of cardiac inflammation on extracellular matrix (ECM) remodeling in patients with HFNEF.
Methods and ResultsWe investigated left ventricular systolic and diastolic function in 20 patients with HFNEF and 8
control patients by conductance catheter methods and echocardiography. Endomyocardial biopsy samples were also
obtained, and ECM proteins as well as cardiac inflammatory cells were investigated. Primary human cardiac fibroblasts
were outgrown from the endomyocardial biopsy samples to investigate the gene expression of ECM proteins after
stimulation with transforming growth factor-. Diastolic dysfunction was present in the HFNEF patients compared with
the control patients. In endomyocardial biopsy samples from HFNEF patients, we found an accumulation of cardiac
collagen, which was accompanied by a decrease in the major collagenase system (matrix metalloproteinase-1) in the
heart. Moreover, a subset of inflammatory cells, which expressed the profibrotic growth factor transforming growth
factor-, could be documented in the HFNEF patients. Stimulation of primary human cardiac fibroblasts from HFNEF
patients with transforming growth factor- resulted in transdifferentiation of fibroblasts to myofibroblasts, which
produced more collagen and decreased the amount of matrix metalloproteinase-1, the major collagenase in the
human heart. A positive correlation between cardiac collagen, as well as the amount of inflammatory cells, and
diastolic dysfunction was evident and suggests a direct influence of inflammation on fibrosis triggering diastolic
dysfunction.
ConclusionsCardiac inflammation contributes to diastolic dysfunction in HFNEF by triggering the accumulation of
ECM. (Circ Heart Fail. 2011;4:44-52.)
Key Words: collagen inflammation remodeling diastolic dysfunction

atients with heart failure with normal ejection fraction

(HFNEF) have increased mortality,1 4 and morbidity has
also been found to be higher compared with patients with HF
and reduced EF.5 One of the underlying mechanisms leading
to the clinical symptomatology in patients with HFNEF is
diastolic function abnormality with increased diastolic stiffness, but nondiastolic function abnormalities with exerciseinduced changes in systolic velocity, chronotropic incompetence, and ventricular-vascular uncoupling have also been
demonstrated to contribute to this disease.6 17 Despite the
growing prevalence of HFNEF in the past 15 years, a disease
affecting about half of the HF population, knowledge about
its molecular mechanisms is still limited, especially because
the pathways leading to HFNEF are not confined to 1
pathology. Intracellular changes with elevated cardiomyocyte

resting tension, as well as a shift in titin isoforms, are

important in patients with severe HFNEF.19,20 Furthermore,
accumulation of cardiac collagen was shown to be present in
this disease and to contribute to the aggravation of diastolic
function.2123 Enhanced endothelial migration of inflammatory cells into the myocardium might influence the development of these changes, especially in regard to changes in the
extracellular matrix (ECM).24,25

Editorial see p 5
Clinical Perspective on p 52
We hypothesized that inflammation is 1 important trigger
of cardiac fibrosis and therefore it plays an important role in
HFNEF. We show herein that increased inflammation triggers collagen accumulation in HFNEF patients and that both

Received December 15, 2009; accepted September 29, 2010.

From the Department of Cardiology and Pneumology (D.W., D.L., M.K., C.Z., K.S., F.E., J.v.S., C.S., A.R., W.P., H.-P.S., C.T.), Charite,
Universititats-Medizin Berlin, Campus Benjamin Franklin, Berlin, Germany, and Department of Cardiology (P.S.), Leiden University Medical Center,
Leiden, The Netherlands.
*Drs Westermann and Lindner contributed equally to this work.
Correspondence to Dirk Westermann, MD, Department of Cardiology, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany.
E-mail dirk.westermann@web.de
2011 American Heart Association, Inc.
Circ Heart Fail is available at http://circheartfailure.ahajournals.org

DOI: 10.1161/CIRCHEARTFAILURE.109.931451

44
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Westermann et al

Study Population
Patients presenting with New York Heart Association class II and III
HF, an EF 50%, and diastolic dysfunction on echocardiography
were evaluated for participation in this study. Persistent atrial
fibrillation and pulmonary diseases were excluded. Significant coronary artery disease or heart valve diseases were ruled out by
angiography. Clinically relevant parvovirus B19 infection with a
genome equivalent 500 was ruled out in all patients included in the
study.26 Twenty patients met the inclusion criteria, were enrolled,
and are referred to as the HFNEF group, with respect to recent
guidelines.27 In addition, 8 patients without signs of congestive HF
who underwent coronary angiography for evaluation of chest pain
were also enrolled in this study and served as controls. In all patients,
endomyocardial biopsy samples were obtained the day after hemodynamic function was analyzed. Cardiac conditions were stable
before catheterization in all patients. All patients gave informed,
written consent. The research protocol was approved by the local
institutional review board.

Pressure-Volume Measurements
and Echocardiography
The conductance catheter was used to assess pressure-volume
measurements in all patients, as recently described in more detail.6 In
brief, left ventricular (LV) diastolic function was characterized by
LV end-diastolic pressure and isovolumetric relaxation (). Furthermore, we calculated the exponential curve fit to the diastolic LV
pressure-volume points during a transient preload reduction to
determine the load-independent diastolic stiffness constant (LV
stiffness constant ). We analyzed LV end-systolic pressure and EF
as a parameter of systolic function. The slope of the end-systolic
pressure-volume relation was calculated as a load-independent parameter for cardiac contractility. Mitral and pulmonary venous
Doppler flow velocities were recorded in the apical 4-chamber view
with a VingMed System FiVe (GE Healthcare, Chalfont St. Giles,
UK) as well as the LV filling index by the ratio of transmitral flow
velocity to annular velocity (E/E lateral), as previously described.7
Right ventricular systolic pressure (in mm Hg) was measured before
the endomyocardial biopsy samples were taken.

Endomyocardial Biopsy
Endomyocardial biopsy samples were obtained from the right side of
the ventricular septum of each patient with use of a flexible bioptome
(Westmed) via the femoral vein approach. Right ventricular systolic
pressure was measured. The tissue pieces were frozen in LN2 and
stored at 70C for subsequent analysis.28 From all patients, 1
endomyocardial biopsy sample was used for immunohistological
staining, and 1 was used for measuring gene expression. (All
measurements were performed once, n20 in HFNEF and n8 in
controls.)

Collagen type IIII

C
% AF

Collagen type I
C
% AF

25

10

P<0.001

P=0.02

15
10
5

2.0

control HFNEF

45

20

control HFNEF

Collag
gen type III
gene e
expression
relative
e to control

Methods

P=0.01

15

Collag
gen type I
expression
gene e
relative
e to control

inflammation and fibrosis are correlated to diastolic dysfunction. We show that inflammatory cells express transforming
growth factor (TGF)-, which directly induces changes in
cardiac fibroblasts as increasing collagen accumulation and
decreasing the degradation system (matrix metalloproteinase
[MMP]-1 and their tissue inhibitor [TIMP]). This was associated with a transdifferentiation of cardiac fibroblasts to
myofibroblasts, known to be a potent contributor of pathological remodelling in diseased tissues. These findings suggest that cardiac inflammation by increased transendothelial
migration is 1 potent trigger of diastolic dysfunction in the
HFNEF population.

Inflammation and Fibrosis in HFNEF

control HFNEF
P=0.03

1.5
1.0
0.5
0.0

control HFNEF

Figure 1. Box-and-whisker plots showing increased levels of collagen types I and III proteins in endomyocardial biopsy samples
from patients with HFNEF compared with controls. Increases in the
collagen I to III ratio as well of collagen type I C-telopeptide were
measured in serum. The mRNA abundance of collagen types I and
type III was increased in HFNEF compared with control subjects.
AF indicates area fractions. *P0.05 vs controls.

Cell Culture
Primary cardiac fibroblasts were obtained by outgrowth from biopsy
specimens from 5 HFNEF patients and were incubated in Iscoves
basal medium (Biochrom AG, Berlin, Germany) containing 10%
human serum, 10% fetal calf serum (FCS), 100 U/mL penicillin
(Biochrom AG), and 100 g/mL streptomycin (Biochrom). The
cardiac fibroblasts were seeded in 24-well plates, and when the
culture was 80% confluent, they were serum-starved in Iscoves
basal medium containing 0.5% FCS (PAA, Colbe, Germany) for 16
hours. They were then stimulated with 5 ng/mL TGF-1 (PeproTech,
Hamburg, Germany) for 6 or 24 hours compared with no stimulation
for 6 or 24 hours. All experiments were done with cells between the
second and fourth passage with n6 wells per experiment for all
time points and all patients. For each patient, a mean was calculated
from the 6 cell culture wells for each experiment (basal conditions
and stimulated for all time points), and the means of all patients are
presented in the figures.
Native fibroblasts were stained with antibodies against CD31,
desmin, vimentin, P4HB, and -smooth muscle actin and compared
with HL1 (experimental atrial cardiomyocytes) as well as with
endothelial cells. The monocyte cell line THP-1 was cultured in
RPMI 1640 medium (PAA) containing 10% FCS (PAA), 100 U/mL
penicillin, and 100 g/mL streptomycin (PAA).

Type I Carboxy-Terminal Telopeptide of Collagen

and Propeptide of Procollagen Type I in Serum
Serum was collected 1 hour before invasive measurements, and type
I carboxy-terminal telopeptide as a breakdown product of collagen
type 1, as well as procollagen type I as a marker of type I collagen
synthesis, was measured as described previously.29

RNA Isolation
Total RNA was extracted from the myocardial biopsy samples or cell
culture wells by the Trizol method (GIBCO BRL, Carlsbad, Calif).

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Circ Heart Fail

20

January 2011
15

P=0.04

10

0.25

control HFNEF

25

P=0.04

% DHE positv
ve nuclei

VCAM-1
% AF

0.20
0 15
0.15
0.10
0.05
0.00

control HFNEF
P=0.01

15

5
0

control HFNEF

The ABI-inventoried TaqMan gene expression assays (each included

forward and reverse primers as well as a fluorescently marked probe)
used for preamplification (from the biopsy samples) and for the
real-time reverse transcriptionpolymerase chain reaction were obtained from Applied Biosystems. Prior real-time reverse transcriptionpolymerase chain reaction cDNA samples (1 to 250 ng) of the
biopsy samples were preamplified with pooled gene expression
assays with the TaqMan PreAmp Master Mix (early access) in a final
volume of 25 L. Quantification of housekeeping CDKN1B transcripts as an internal control for the amount and quality of cDNA was
performed for all samples. Gene expression for collagen types I and
III was analyzed from cardiac biopsy samples (primers from Applied
Biosystems). Data for cell culture were also normalized to human
CDKN1B mRNA levels as an endogenous control (unaffected by
TGF- treatment) and are expressed relative to untreated controls
according to the formula 2CT.

TGF- ELISA
The THP-1 cells were starved in Iscoves medium containing 0.5%
FCS (PAA), 100 U/mL penicillin, and 100 g/mL streptomycin
(PAA) 16 hours before the experiment. THP-1 cells (3 millions [ml])
were treated with 100 ng/mL phorbol 12-myristate 13-acetate
80

control HFNEF

50

P=0.002

60

CD
D45+ cells / mm
m

CD
D11a+ cells / mm
m

CD3+ cells / mm

10

Figure 2. Decreases in protein levels of MMP-1

and increases in levels of MMP-2 and TIMP-1
were found in endomyocardial biopsy samples
from patients with HFNEF compared with controls. Increased oxidative stress by DHE staining was documented in HFNEF compared with
control subjects. The adhesion molecule
VCAM-1 was also increased in HFNEF compared with control subjects. AF indicates area
fractions. *P0.05 vs controls.

10

Real-Time Reverse TranscriptionPolymerase

Chain Reaction

20

control HFNEF

20

control HFNEF

P<0.001

20
10

Additional purification was performed with the ChargeSwitch total

RNA cell kit (Invitrogen, Karlsruhe, Germany). The yield of purified
total RNA was analyzed by checking the UV absorbance at 260 nm
on a NanoDrop ND-1000 (Agilent Technologies, Boeblingen, Germany) spectrophotometer.

30

P=0.01

30

10

TIMP-1
% AF

MMP-1
% AF

15

40

P=0.02

MMP-2
% AF

46

40
20
0

control HFNEF

(Sigma-Aldrich) for 12, 24, or 48 hours. The cell culture supernatant

was then used for measuring the total TGF-1 concentration with the
TGF1 Emax immunoassay system (Promega). The cell culture
supernatant was first diluted 1:5 in Dulbeccos phosphate-buffered
saline (PAA) and then acidified with HCl to pH 2.6 for 15 minutes,
followed by neutralization with NaOH before using the sample for
the TGF1 Emax immunoassay system.

Histology
Immunohistological staining was performed by standard techniques.
The antibodies were purchased from Chemicon (anti-MMP1, CD3,
CD11a, CD45, vascular cell adhesion molecule [VCAM]-1, and
collagen types I and III) and Calbiochem (anti-TIMP-1 and anti
MMP-2). Immunohistochemical staining images were quantified by
digital image analysis. For double immunofluorescence, we used
Tissue Tec embedded cryosections. The following primary antibodies were used: mouse anti-sarcomere actin (1:50, Santa Cruz) and
mouseantiTGF- (1:50, Serotec). The secondary antibodies were
labeled with fluorescein isothiocyanate and tetramethylrhodamine
isothiocyanate (Sigma, St. Louis, Mo). Nuclei were visualized with
4,6-diamidino-2-phenylindole (Sigma). Slides were embedded in
fluorescent mounting medium (DakoCytomation, Glostrup, Denmark), coverslipped, and analyzed by fluorescence microscopy
(Zeiss, Jena, Germany). Sections were incubated with 10 mol/L
dihydroethidium to detect O2 and visualized by fluorescence
microscopy, whereby DHE-positive nuclei were counted with respect to the number of total nuclei.

Statistical Analysis
Data are shown in box-and-whisker plots (Figures 1 through 3) and
as meanSEM (Figures 4 and 5). For comparison of the HFNEF
P<0.001

40

Figure 3. Increased numbers of CD3, CD11,

and CD45 cells were found in endomyocardial
biopsy samples from patients with HFNEF compared with controls. *P0.05 vs controls.

30
20
10
0

control HFNEF

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Westermann et al

Inflammation and Fibrosis in HFNEF

47

Figure 4. A, Representative histologic image with double staining of CD11 cells with the profibrotic growth factor TGF-. B, In vitro
experiments with phorbol 12-myristate 13-acetateactivated monocytes (THP-1 cells) showed increased production of TGF- mRNA
and protein levels of TGF- in a time-dependent manner. *P0.05 vs individual controls.

group with the control group, the nonparametric MannWhitney U

test was used for data that were not normally distributed. Fishers
exact test was used to analyze categorical variables. A probability
value 0.05 was considered statistically significant. Data were
analyzed with Graphpad 5.01 (PRISM, San Diego, Calif) and SPSS
(version 15.0; SPSS Inc, Chicago, Ill).

Results
Patient Demographics
Patient characteristics are summarized in Table 1. There were
no significant differences between HFNEF patients and
controls with respect to age, sex, or body surface area. The
prevalence of hypertension and diabetes mellitus was not
statistically different between the groups (Table 1).

Hemodynamic Data
Systolic function was not changed in HFNEF compared with
controls. In contrast, diastolic dysfunction was evident in the
HFNEF group, with an increased LV stiffness constant ,
increased LV end-diastolic pressure, and prolonged isovolumetric relaxation. Echocardiography revealed that the cardiac
dimensions of both groups were not significantly different.
Diastolic dysfunction with increased E/E lateral was documented in the HFNEF group. Moreover, significant cardiac
hypertrophy was documented in HFNEF compared with
control subjects (Table 2). Right ventricular systolic pressure,
as 1 parameter of pulmonary hypertension, was not significantly different in HFNEF and controls (29.7; range, 24 to
33 mm Hg, vs 25; range, 21 to 28.5 mm Hg; P0.4).

Figure 5. In vitro cell culture experiments of primary cardiac fibroblasts

stimulated with TGF- for 6 or 24 hours.
Increased gene expression of -smooth
muscle actin (a-SMA), connective tissue
growth factor (CTGF), and collagen type
I (Col I) were found in stimulated cells
compared with unstimulated cells.
MMP-1 was decreased after stimulation
with TGF-. MMP-2 was increased after
stimulation. *P0.05 compared to the
relative controls.

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48

Circ Heart Fail

Table 1.

January 2011

Characteristics of the HFNEF and Control Groups

Control
(n8)

HFNEF
(n20)

P Value

56 (50 69)

60 (43 68)

0.21*

12

Characteristics
Age, y
NYHA class II
NYHA class III

51 (2867)

573 (389949)

-blockers

14

0.018

ACE inhibitors/ARBs

17

0.0048

Ca2 channel blockers

0.536

Diuretics

16

0.011

Hypertension

12

0.20

Diabetes mellitus

0.14

NT-pro-BNP, pg/mL

0.001*

Type I carboxy-terminal telopeptide, as 1 serum marker for

collagen degradation, was increased in the sera of HFNEF
patients compared with controls (meanSEM, 5.92.1 vs
2.31.4 g/L; P0.025). Procollagen type I, a serum marker
for collagen production, was also increased in the HFNEF
group compared with controls (meanSEM, 128.926 vs
65.121 g/L; P0.018).

Medications

Concomitant diseases

NYHA indicates New York Heart Association; NT-pro-BNP, NT-pro-brain

natriuretic peptide; ACE, angiotensin-converting enzyme; and ARB, angiotensin
receptor blocker. Patient characteristics and data are shown as median
(25%75% percentile).
*t test.
Fishers exact test.

Inflammation
The inflammatory cells marked by CD3, CD11a, and CD45
were all increased in the cardiac tissues of the HFNEF
patients compared with controls (Figure 3). Moreover, the
adhesion molecule VCAM-1 was increased in HFNEF compared with control patients (Figure 2). In double staining,
inflammatory cells revealed a secretion of the profibrotic
growth factor TGF- (Figure 4). More cells (mostly cardiomyocytes and endothelial cells) in the HFNEF group were
positive after DHE staining as a marker of oxygen radical
production in comparison with controls (Figure 2). No change
in systemic inflammation as measured by C-reactive protein
was found between the groups (1.30.5 vs 1.10.3 mg/dL;
PNS).

Extracellular Matrix
Collagen type I and III expression in the cardiac biopsy
samples was significantly higher in patients with HFNEF
than in controls. Moreover, mRNA abundance of both collagen subtypes was increased in HFNEF patients (Figure 1).
The ratio of collagen type I to type III was increased in
HFNEF patients. TGF- mRNA expression in the biopsy
samples was increased in HFNEF patients compared with
controls (59% compared with controls, P0.004). Histologic
expression levels of cardiac MMP-1, the major human
collagenase, were decreased in HFNEF, whereas TIMP-1 was
increased in HFNEF individuals compared with controls
(Figure 2).
Table 2.

Echocardiographic and Hemodynamic Results

Control

HFNEF

LVEDD

47 (4249)

Septum

9.8 (8.310.2)

12.3 (10.513.6)

0.001*

Posterior wall

9.2 (8.69.5)

11.4 (10.212.2)

0.001*

Tissue Doppler E/E

(lateral)

6.1 (4.77.5)

13.9 (11.417.4)

0.011*

0.573*

Pressure-volume loops
LVP, mm Hg
dP/dtmax, mm Hg/s
ESPVR, mm Hg/mL
LVEDP, mm Hg

, ms
Diastolic stiffness

131 (117145)

136 (124159)

1783 (15092106)

1882 (16392117)

1.1 (0.81.3)
6 (49)
48 (4049)
0.01 (0.0060.04)

0.412
0.535

1.2 (11.5)

0.386

16 (1424)

0.001*

64 (6378)

0.001*

0.06 (0.030.12)

After stimulation with TGF- for 6 or 24 hours, connective

tissue growth factor and collagen type 1A1 mRNAs were
upregulated compared with controls. The transdifferentiation
to myofibroblasts, as suggested by -smooth muscle actin
mRNA expression, was observed after 24 hours of stimulation with TGF-. MMP-1 was not increased after 6 or 24
hours, but MMP-2 was upregulated after 24 hours. With an
increased TIMP-1 expression, the MMP-1 to TIMP-1 ratio as
1 indicator of MMP-1 activity was downregulated after
stimulation with TGF- for 6 or 24 hours (Figure 5).

THP-1 Cell Culture

P Values

Chamber dimensions,
mm
46 (4050)

Fibroblast Cell Culture

0.001*

LVEDD, LV end-diastolic diameter; LVP, LV pressure; ESPVR, end-systolic

pressure volume relation; LVEDP, LV end-diastolic pressure; and , isovolumetric relaxation time. Data are shown as median (25%75% percentile).
*t test.

Activated THP-1 cells produced TGF- mRNA compared

with their nonactivated controls. This was followed by a
time-dependent production of TGF- protein, which showed
significantly increased protein levels 24 hours after stimulation of the THP-1 monocytes (Figure 4B).

Discussion
The salient finding of the current study is that cardiac
inflammatory cells documented in endomyocardial biopsy
samples from patients with HFNEF induce extracellular
remodelling with increased accumulation of collagen.
TGF-, excreted by inflammatory cells, is 1 potent stimulus
for the regulatory changes of the ECM, as shown in cell
culture experiments with primary cardiac fibroblasts derived
from endomyocardial biopsy samples of patients with HFNEF.
These changes contribute to diastolic dysfunction, which is 1
of the underlying pathologies of HFNEF, which was documented by a correlation between collagen (as well as inflammatory cells) and diastolic dysfunction.

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Westermann et al
The molecular changes leading to diastolic dysfunction are
still debated, and different pathways can affect the pathology
of HFNEF. Altered isoform expression of the giant muscle
protein titin can determine the elastic properties of the heart.19
Moreover, increased myocyte tension, which can be prevented by experimental phosphorylation of titin, contributes
to diastolic dysfunction in patients with severe HFNEF.20,30
Additional modulatory effects on titin stiffness may arise
from disulfide bonding under oxidant stress also affecting
LV compliance.31 Nevertheless, the steep part of the
diastolic pressure-volume relation is mainly modified by
the ECM, as can be observed when comparing stretch
lengths between whole-muscle stripes and single myocytes,32 suggesting that excess collagen might further
aggravate diastolic dysfunction.
Cardiac collagen is a stable protein with a low turnover (80
to 120 days),33 but its balance can be disrupted in pathological
conditions. Next to ischemia, which may lead to replacing
fibrosis, for example, increased wall stress, angiotensin II,
and TGF- may induce profibrotic processes leading to
pathological tissue fibrosis.34 The tensile strength of collagen
type 1 approximates that of steel. Therefore, LV chamber
stiffness in vivo will be affected by the amount of cardiac
ECM.35 In this study, we showed that total cardiac collagen
content was increased in patients with HFNEF, in agreement
with other work that showed increased fibrosis in this
disease.19,21,36 Furthermore, not only was collagen type I
found to be increased in this study population, but also there
was a change in the collagen type I to III ratio in favor of
the stiffer collagen type I, similar to that in patients with
systolic HF.37 In addition to increased protein levels of
collagen types I and III, their mRNA abundance was also
increased in endomyocardial biopsy samples from patients
with HFNEF compared with controls. Another marker of
excessive collagen production is type I carboxy-terminal
telopeptide, a degradation product of collagen with increased collagen turnover together with propeptide of
procollagen type I, a serum marker of collagen production,
both of which were increased in the HFNEF group.
Oxidative stress was increased in HFNEF patients, as DHE
staining revealed, which might be the result of increased
LV stiffness.
Multiple studies have shown that 1 of the best-known
inducers of collagen production is the profibrotic growth
factor TGF-. It also has profound effects on ECM
homeostasis, in part through its ability to alter the balance
between MMPs and their TIMP inhibitors. The endogenous collagen degradation system is regulated by increased
activity of MMPs overcoming their tissue inhibitors.38
Nevertheless, there are different MMPs, and the substrate
affinity of those proteases is different.39 MMP-1 (interstitial collagenase) is known to degrade collagen fibers, and
therefore it likely favors collagen degradation. Through
the activity of the activator protein-1 transcription factor,
TGF- can, on one hand, repress MMP-1 gene expression40 and, on the other hand, increase TIMP-1 expression.
Congruent with these ex vivo data, we have show an
upregulation of TIMP-1 protein and a downregulation of
MMP-1 protein levels in the biopsy samples from HFNEF

Inflammation and Fibrosis in HFNEF

49

patients, which leads to a significant decrease in the

MMP-1TIMP-1 ratio. This inhibition of the collagen
degradation system could be 1 mechanism contributing to
the accumulation of ECM in HFNEF patients and the
initiation of diastolic dysfunction over a longer time
period.41 Interestingly, Lopez and colleagues22 showed that
this ratio was increased in patients with systolic HF,
whereas it was unchanged in their hypertensive HF group.
Whether this process is dynamic or changes with time, or
whether this mechanism represents a distinct difference
between both HF subtypes remains to be clarified.
Furthermore, and in contrast to the activator protein-1
mediated downregulation of MMP-1, we found increased
levels of MMP-2 (controlled by activator protein-2), which is
a known gelatinase and has substrate affinity for denatured
fibrillar collagen as well as for the basement membrane.42
Some studies have described MMP-2 serum levels in patients
with hypertensive and diastolic HF, but the results are
inconsistent, with some studies showing an increase,43 and
others showing no difference or even a decrease in MMP-2.33
Recently, increased levels of MMP-2 were shown to predict
HF in patients with diastolic dysfunction and hypertension.
In that study, MMP-2 was a better prognostic marker than
the well-known HF biomarker brain natriuretic peptide.29
Several experimental studies in MMP-2 knockout animals
have helped to advance understanding of the molecular
function of MMP-2. Matsumura and colleagues44 showed
in MMP-2 knockout mice a decrease in invading inflammatory cells and a decrease in LV rupture after myocardial
infarction. Those authors demonstrated that destruction of
basement membrane proteins facilitates the transendothelial migration of immunocompetent cells, thereby triggering cardiac inflammation.
In light of such findings, we investigated the number of
inflammatory cells in our patients and showed that HFNEF
is associated with increased cardiac inflammation, with
high numbers of CD3, CD11a, and CD45 cells.
Moreover, the adhesion molecule VCAM-1, which attracts
immunocompetent cells to the endothelium and initiates
transendothelial migration, was also increased in the
HFNEF group. This result is especially interesting, because VCAM-1 is upregulated by angiotensin II, which
may be increased in regard to known risk factors like
hypertension and diabetes mellitus in HFNEF. Recently, it
was shown that immunocompetent cells like T-cells
(CD3) can indeed alter tissue remodelling in vitro,45 and
we have shown that cardiac inflammation is associated
with excessive collagen accumulation in experimental
diabetic cardiomyopathy in 1 animal model of HFNEF.25
There is experimental and clinical evidence that inflammatory cells might modulate cardiac function in HF with
reduced46,47 and normal48,49 EF. The direct effects of these
cells are still under debate, but it has been suggested that
increased inflammation is associated with the development
of systolic HF by distinct changes in the ratio of MMP to
TIMP.50,51 In line with these data, we have demonstrated
herein that these inflammatory cells express TGF- in
cardiac tissue, as shown by immunohistochemical double
staining. Moreover, THP-1 cells, when they become acti-

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Circ Heart Fail

January 2011

vated, which is 1 hallmark of transendothelial migration,

produce TGF- on the mRNA and protein level, as
evidenced by in vitro studies of phorbol 12-myristate
13-acetateactivated THP-1 cells. In addition, TGF- gene
expression was increased in the HFNEF biopsy samples.
To test the direct effect of TGF- expressed by inflammatory cells in this study, we performed experiments in a
cell culture system with primary human cardiac fibroblasts
derived from endomyocardial biopsy samples of HFNEF
patients. After stimulation with TGF-, fibroblasts expressed increased amounts of -smooth muscle actin, 1
marker for their transdifferentiation into pathologically
activated myofibroblasts. Connective tissue growth factor
was concomitantly increased, leading to higher collagen
gene expression after 6 and 24 hours, which explains the
collagen accumulation found in the endomyocardial biopsy
samples. On the other hand, the degradation system of
collagen, especially the MMP-1 to TIMP-1 ratio, was
downregulated after stimulation with TGF- in the cell
culture system, which is another explanation for the
extensive fibrosis seen in HFNEF hearts. MMP-2, in line
with its known function in degrading the basement membrane to allow for easier transendothelial migration of
inflammatory cells into cardiac tissue, was upregulated
after TGF- stimulation. Interestingly, TGF- gene expression was increased after stimulation, which suggests
that the already transdifferentiated myofibroblasts might
have induced further activation of fibroblasts and therefore
enhanced the profibrotic process in a paracrine fashion.
Viral agents are 1 possible inducer of cardiac inflammation. The parvovirus B19, which is associated with
endothelial and diastolic dysfunction,28 is known to cause
myocarditis. Nevertheless, it was recently shown that in
patients with dilated cardiomyopathy, a viral load of only
500 genome equivalents is a clinically relevant threshold
for the maintenance of myocardial inflammation.26 Because this extent of viral load was ruled out in all patients,
the exact mechanism of cardiac inflammation has still to be
determined, although animal models of HF are often
associated with cardiac inflammation.24 Recently, it was
shown52 that activated myofibroblasts also produce chemokines to fuel the inflammatory process, which might
induce a vicious circle of inflammation triggering fibrosis
and diastolic dysfunction. If hemodynamic changes in
HFNEF induce proinflammatory changes to start cardiac
inflammation, these have to be determined in detail in
further studies.
We suggest that small numbers of invading inflammatory
cells stimulate cardiac fibrosis by expressing TGF- and
inducing a pathological transdifferentiation from fibroblasts
to myofibroblasts. This will not only stimulate gene expression of collagen but also induce a decline in MMP-1 activity
and an increase in MMP-2 mRNA abundance, as well as
paracrine TGF- production. Increased stiffness due to ECM
accumulation will increase oxidative stress, suggested to be
associated with increased endothelial activation. Together
with increased MMP-2, which has been suggested to disrupt
the basal membrane, this might induce a vicious circle

fuelling inflammation leading to fibrosis and ultimately, to

progression of the disease.

Conclusions
In this study, HFNEF was characterized by an increase in
cardiac inflammation. This inflammatory process triggers
cardiac collagen accumulation by inducing collagen gene
expression and inhibiting the cardiac degradation system.
Inhibiting the transendothelial migration of inflammatory
cells into cardiac tissue might be a future therapeutic concept
in HFNEF.

Sources of Funding
This study was supported by the Deutsche Forschungsgemeinschaft
(SFB-TR-19, A2, and B5) and by the European Commission,
Research Directorate General, FP7-Health-2010, MEDIA (261409),
Brussels, Belgium.

Disclosures
None.

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CLINICAL PERSPECTIVE

Heart failure with normal ejection fraction (HFNEF) can be diagnosed in 50% of patients with the clinical syndrome of
HF. Nevertheless, the HFNEF syndrome seems to be heterogeneous and therefore will present with different pathologies
leading to the disease. It is assumed that an accumulation of cardiac collagen is 1 important mediator in the development
of HFNEF. We show here that increased transendothelial migration of inflammatory cells triggers a profibrotic phenotype
by activating fibroblasts into myofibroblasts in cardiac tissue. Activated myofibroblasts, characterized by -smooth muscle
actin, are well known for excessive collagen production in other diseased tissues and express significantly more collagen
compared with normal cardiac fibroblasts. Moreover, inflammatory cells reduced the expression of the collagen
degradation system, the matrix metalloproteinases, again promoting cardiac fibrosis as 1 trigger for a stiff and
noncompliant ventricle. This transdifferentiation was induced by transforming growth factor-, which was expressed by
inflammatory monocytes in the heart. Interestingly, we document here that these inflammatory cells can be found more
frequently in cardiac tissue of the HFNEF group than in the control group. Therefore, continuous low-grade inflammation
might be 1 key player in the development and/or progression of HFNEF. This would make anti-inflammatory strategies
inhibiting transendothelial migration of cells into the cardiac tissue an interesting approach in treating HFNEF, a disease
plagued by limited data.

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