Download Elevated circulating cardiotrophin-1 in heart failure

Clinical Science (2000) 99, 83–88 (Printed in Great Britain)
Elevated circulating cardiotrophin-1 in heart
failure: relationship with parameters of left
ventricular systolic dysfunction
S. TALWAR, I. B. SQUIRE, P. F. DOWNIE, R. J. O’BRIEN, J. E. DAVIES and L. L. NG
Department of Medicine and Therapeutics, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary,
Leicester LE2 7LX, U.K.
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Cardiotrophin-1 (CT-1) is a cytokine that has been implicated as a factor involved in myocardial
remodelling. The objective of the present study was to establish the relationship between
circulating levels of CT-1 and measures of left ventricular size and systolic function in patients
with heart failure. We recruited 15 normal subjects [six male ; median age 60 years (range 30–79
years)] and 15 patients [11 male ; median age 66 years (range 43–84 years)] with a clinical diagnosis
of heart failure and echocardiographic left ventricular systolic dysfunction (LVSD). Echocardiographic variables (left ventricular wall motion index, end-diastolic and -systolic volumes, stroke
volume, fractional shortening) and plasma CT-1 levels were determined. In patients with LVSD
[median wall motion index 0.6 (range 0.3–1.4)], CT-1 was elevated [median 110.4 fmol/ml (range
33–516 fmol/ml)] compared with controls [wall motion index 2 in all cases ; median CT-1 level
34.2 fmol/ml (range 6.9–54.1 fmol/ml) ; P 0.0001]. Log CT-1 was correlated with log wall
motion index (r l k0.76, P 0.0001), log left ventricular end-systolic volume (r l 0.54,
P 0.05), stroke volume (r l k0.60, P l 0.007) and log fractional shortening (r l k0.70,
P l 0.001). In a multivariate model of the predictors of log wall motion index, the only significant
predictor was log CT-1 (R2 l 56 %, P l 0.006). This is the first assessment of the relationship
between plasma CT-1 levels and the degree of LVSD in humans, and demonstrates that CT-1 is
elevated in heart failure in relation to the severity of LVSD.
INTRODUCTION
The recently cloned novel cytokine cardiotrophin-1 (CT1) is a member of the family of interleukin-6-related
cytokines that bind to glycoprotein 130 (gp130)
receptors. The family includes interleukin-6, interleukin11, ciliary neurotrophic factor, leukaemia inhibitory
factor (LIF) and oncostatin M [1]. CT-1 is expressed at
high levels in the myocardium during cardiogenesis,
promoting the proliferation and survival of embryonic
cardiomyocytes [1–3].
CT-1 induces cardiac myocyte hypertrophy [1], adding
sarcomeres in series rather than in parallel and leading to
increased cardiac myocyte size due to an increase in cell
length, with little change in width [2]. CT-1 binds to the
LIF receptor, leading to tyrosine phosphorylation of
the gp130\LIF receptor heterodimer [2,3]. Signalling
cascades involved include the Janus kinase (JAK)\signal
Key words : cardiotrophin-1, cytokines, echocardiography, heart failure.
Abbreviations : ACE, angiotensin-converting enzyme ; BNP, brain (B-type) natriuretic peptide ; CT-1, cardiotrophin-1 ; EDV, left
ventricular end-diastolic volume ; ESV, left ventricular end-systolic volume ; FS, fractional shortening ; gp130, glycoprotein 130 ;
JAK, Janus kinase ; LIF, leukaemia inhibitory factor ; LVID, left ventricular internal dimensions ; LVIDd, LVID at end-diastole ;
LVIDs, LVID at end-systole ; LVSD, left ventricular systolic dysfunction ; STAT, signal transducer and activation of transcription ;
SV, stroke volume ; WMI, wall motion index.
Correspondence : Dr L. L. Ng (e-mail lln1!le.ac.uk).
# 2000 The Biochemical Society and the Medical Research Society
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S. Talwar and others
transducer and activation of transcription 3 (STAT3)
pathway [4].
In addition to its effects on cardiomyocyte hypertrophy, CT-1 has also been shown to prevent myocyte
apoptosis via a pathway dependent on activation of a
mitogen-activated protein kinase [5]. Further evidence of
a cardiac cytoprotective effect was evident in experiments
where CT-1 was shown to induce accumulation of heatshock protein in cultured cardiac cells, protecting them
from ischaemic stress [6]. The hypertrophic response of
cardiac myocytes to CT-1 is dependent on the JAK\
STAT pathway, whereas the anti-apoptotic effects of
CT-1 employ signalling via the mitogen-activated protein
kinase pathway.
In experimental animals, the acute haemodynamic
effects of CT-1 include an increase in cardiac output via
an increase in heart rate, and a decrease in systemic
vascular resistance without a direct positive inotropic
effect [7]. In addition, CT-1 stimulates the production of
brain (B-type) natriuretic peptide (BNP) at a transcriptional level by a mechanism that is distinct from the
effect of endothelin-1 on cultured neonatal rat cardiomyocytes [8,9].
Increased expression of CT-1 occurs in a rat model of
acute myocardial infarction, and this may relate to a role
for CT-1 in the pathogenesis of ventricular remodelling
[10]. In addition, overexpression of cardiac CT-1 and
gp130 occurs during experimental heart failure [11].
Cardiomyocytes from humans with ischaemic cardiomyopathy are about 50 % longer than normal, suggesting
the addition of sarcomeres in series in left ventricular
systolic dysfunction (LVSD) [12]. As noted above, CT-1
induces cardiac myocyte hypertrophy via the addition of
sarcomeres in series rather than in parallel and via
increased cardiac myocyte length with little change in
width. These data suggest a possible role for CT-1
in ventricular remodelling following acute myocardial
infarction in humans, and also in patients with established
heart failure. There are currently no data on circulating
CT-1 levels in subjects with LVSD.
We hypothesized that CT-1 levels would be raised in
subjects with LVSD. We thus examined whether CT-1 is
detectable in the plasma of normal humans, whether
plasma levels are elevated in patients with heart failure,
and the relationship between plasma CT-1 levels and
measures of LVSD. We used a competitive immunoluminometric assay for detecting circulating CT-1 using
techniques developed for the measurement of plasma
levels of N-terminal proBNP [13].
[11 male ; median age 66 years (range 43–84 years)] and 15
normal subjects [six male ; median age 60 years (range
30–79 years)]. Normal subjects were nine individuals
[four male ; median age 40 years (range 30–79 years)] with
normal echocardiographic examination and six healthy
volunteers [two male ; median age 63 years (range 48–79
years)]. None of the normal subjects was taking any
regular prescribed medication and none had any history
of cardiovascular disease. Of the patients with LVSD, 10
were receiving a loop diuretic, seven an angiotensinconverting enzyme (ACE) inhibitor, two digoxin and
one a β-blocker.
Patients and subjects with normal echocardiographic examination were recruited during attendance
for a routine outpatient echocardiographic examination.
Healthy volunteers were the spouses or partners of patients. The study was approved by the Leicester Health
Authority Ethics Committee. All subjects gave written
informed consent to participation.
Echocardiography
Echocardiography was performed using a Hewlett
Packard Sonos 1500 imaging system. Wall motion index
(WMI) was calculated using a validated nine-segment
model [14,15], blind to patient details. LVSD was defined
as WMI 1.4, corresponding to a left ventricular ejection
fraction of 35 %. Left ventricular chamber dimensions
were obtained at the tip of the mitral valve. Interventricular septal thickness and posterior wall thickness
were measured at end-diastole. Left ventricular internal
dimensions (LVID) were obtained at end-diastole
(LVIDd) and at end-systole (LVIDs). Fractional shortening (FS) was calculated as the percentage change in the
LVID between systole and diastole. Left ventricular
volumes were calculated according to the formula of
Teicholz et al. [16]. Left ventricular stroke volume
(SV) was defined as the left ventricular end-diastolic
volume (EDV) minus the left ventricular end-systolic volume (ESV).
Blood sampling
Blood sampling was carried out following echocardiographic examination. Following 30–45 min of supine
rest, 20 ml of blood was transferred to pre-chilled tubes
containing 500 units\ml aprotinin (Trasylol ; Bayer UK,
Newbury, U.K.) and EDTA (1.5 mg\ml). Plasma was
separated immediately and stored at k70 mC until assay
for CT-1 within 2 months.
Assay for CT-1
METHODS
Subjects
We studied 15 consecutive patients with a clinical
diagnosis of heart failure and echocardiographic LVSD
# 2000 The Biochemical Society and the Medical Research Society
Details of the immunoluminometric competitive binding
assay for CT-1 have been described in full previously
[17]. Briefly, stored plasma samples were extracted using
C extraction cartridges and redissolved in assay buffer
")
for measurement of CT-1. The CT-1 peptide was
Cardiotrophin and heart failure
synthesized in the Medical Research Council Toxicology
Unit, Leicester University, and conjugated to keyholelimpet haemocyanin to immunize rabbits by monthly
subcutaneous injections. An in-house polyclonal antibody to amino acids 105–120 of the CT-1 sequence was
developed and a methyl acridinium ester was used to
label the peptide. Plasma extracts were incubated with the
antibody before addition of the tracer. Immune complexes were recovered with paramagnetic particle
immobilized goat anti-rabbit globulin and, after extensive
washes, chemiluminescence was measured by sequential
injections of acidified H O (0.1 M HNO ) and 0.25 M
# #
$
NaOH. Peptide recovery using the C column plasma
")
extraction process was 76.2p3 %.
Assays were performed using a Berthold Autolumat
LB953 luminometer. Within- and between-assay coefficients of variation were 6.2 % and 10.3 % respectively.
Cross-reactivity with other peptides (i.e. atrial natriuretic
peptide, BNP, N-terminal proBNP, interleukin-6 and
LIF) known to be elevated in heart failure was 0.1 % in
all cases. CT-1 levels were determined blind to patient
details and echocardiographic findings. Each CT-1 value
represents the mean of duplicate measurements.
Table 1 Comparative echocardiographic data for patients
with LVSD and control subjects
Abbreviations : IVST, interventricular septal thickness ; PWT, posterior wall
thickness. Values are median (range). Significance of differences compared with
patients with LVSD : *P 0.05 ; **P 0.005 ; ***P 0.001.
Parameter
Patients with LVSD
Controls
WMI
FS (%)
IVST (cm)
PWT (cm)
LVIDd (cm)
LVIDs (cm)
Left atrial diameter (cm)
ESV (ml)
EDV (ml)
SV (ml)
0.6 (0.3–1.4)
17.2 (11.4–25)
1.15 (0.91–2.04)
1.12 (0.91–2.04)
5.03 (3.44–7.41)
3.93 (2.84–6.16)
3.8 (1.92–8.37)
67.1 (30.6–191)
120.5 (48.8–290)
49 (18.2–99)
2***†
46.9 (30.2–59)***
0.91 (0.6–1.02)*
0.9 (0.58–1.02)*
5.12 (4.3–5.82)
2.8 (1.98–3.0)**
3.5 (2.8–4.47)
29.6 (12.4–35)**
125 (83.1–168)
99.1 (48.1–138)*
† All control subjects had a WMI of 2.
Statistical analyses
All statistical analyses were carried out using the software
package Minitab (Minitab Inc.). All results are expressed
as median (range). Values of CT-1, WMI, interventricular
septal thickness, posterior wall thickness, FS, ESV, EDV
and SV were normalized by logarithmic transformation
prior to analyses. For the categorical variables clinical
heart failure, current diuretic use and current ACE
inhibitor use, 95 % confidence intervals were calculated
for the difference in plasma CT-1 values between patients
with and without the variable of interest. Comparisons
between patients with LVSD and control subjects were
made using the Mann–Whitney U-test for non-parametric data.
Figure 1 Plasma CT-1 concentrations in patients with LVSD
and in control subjects
Logarithmic transformation has been carried out. Median values are shown as
horizontal lines.
RESULTS
Echocardiographic data obtained from LVSD subjects
and controls are reported in Table 1. Five patients with
LVSD were categorized within New York Heart Association class II, eight patients within class III and two
patients within class IV. Median WMI in the 15 patients
with LVSD was 0.6 (range 0.3–1.4). Plasma CT-1 levels
were elevated in patients with LVSD (median 110.4,
range 33–516 fmol\ml) compared with control subjects
(median 34.2, range 6.9–54.1 fmol\ml ; P 0.0001) (Figure 1).
Positive correlations were apparent between log
plasma CT-1 level and a number of echocardiographic
indices : log WMI (r l k0.76, P 0.0001) (Figure 2), log
Figure 2 Correlation between log CT-1 and log WMI in
patients with LVSD and in control subjects
# 2000 The Biochemical Society and the Medical Research Society
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S. Talwar and others
Figure 3 Correlation between log CT-1 and log FS in patients
with LVSD and in control subjects
respectively (95 % confidence interval for difference
between medians k75 to 32 ; not significant).
We considered age, gender, history of ischaemic heart
disease or hypertension, current diuretic or ACE inhibitor use, presence of clinical left ventricular failure and
log CT-1 in a multivariate model of the predictors of log
WMI. The only significant predictor was log CT-1 (P l
0.006). On best-subset analysis the strongest correlate
with log WMI was log CT-1 (R# l 55.5 %).
We performed further analysis, and looked at determinants of circulating CT-1 levels in our patients with
heart failure. In a multiple-regression model the determinants of CT-1 were log WMI (P 0.005), the presence
of a history of hypertension (P l 0.006), the age of the
patient (P 0.05), log interventricular septal thickness (P
l 0.009), LVIDd (P 0.05), LVIDs (P 0.05), log
posterior wall thickness (P l 0.007), log ESV (P 0.05)
and log EDV (P 0.05). In a best-subset analysis log
WMI, history of hypertension, LVIDs and log ESV
accounted for 32.6 % of the variance in plasma CT-1
levels (R# l 32.6 %).
DISCUSSION
Figure 4 Correlation between log CT-1 and log ESV in
patients with LVSD and in control subjects
FS (r l k0.70, P 0.001) (Figure 3), LVIDs (r l 0.53, P
0.05), log ESV (r l 0.54, P 0.05) (Figure 4) and log
SV (r l k0.60, P l 0.007). In addition, log WMI correlated with log FS (r l 0.70, P l 0.001).
Although in the patient group plasma CT-1 levels
tended to be higher in those with clinical heart failure
than in those without, and in those receiving diuretic or
ACE inhibitor therapy, no statistically significant difference could be demonstrated between patients with any
one of these categorical variables and those without.
Median CT-1 levels in those with (n l 11) and without (n
l 4) clinical heart failure were 111 and 78 fmol\ml
respectively (95 % confidence interval for difference
between medians k71 to 39 ; not significant) ; in those
with (n l 8) and without (n l 7) current diuretic use
were 92 and 58 fmol\ml respectively (95 % confidence
interval for difference between medians k79 to 39 ; not
significant) ; and in those with (n l 2) and without (n l
13) current ACE inhibitor use were 111 and 71 fmol\ml
# 2000 The Biochemical Society and the Medical Research Society
Long-standing exposure to hypertension or haemodynamic stress secondary to ischaemic injury can activate
a distinct form of myocardial hypertrophy resulting in
cardiac dilatation. This process of remodelling involves
the addition of new myocytes in series and results in
irreversible loss of myocardial function. The identification of stimuli that initiate and maintain the processes
of cardiac hypertrophy and remodelling, and eventually
heart failure, remains a major pursuit in cardiac molecular
biology and medicine. This is the first report of elevated
plasma CT-1 levels in subjects with LVSD. While the
exact role of CT-1 in LVSD and its relationship with
ventricular remodelling remain to be defined, related
cytokines such as tumour necrosis factor-α and certain
interleukins are capable of modulating cardiovascular
function by a variety of mechanisms, including the
promotion of left ventricular remodelling [18,19].
We have demonstrated a strong correlation between
the magnitude of the rise in plasma CT-1 levels and the
severity of LVSD. Plasma CT-1 levels were particularly
correlated with two measures of left ventricular systolic
function, namely WMI and FS. Furthermore, we have
demonstrated CT-1 level to be a significant predictor of
WMI in a multivariate model. Whether measurement of
CT-1 levels in patients with known or suspected LVSD
can be of clinical use in terms of diagnosis or monitoring
of disease progress remains to be tested in larger
prospective studies. In this respect, the relative merits of
CT-1 in comparison with other neurohormones such as
BNP will be of interest.
Cardiotrophin and heart failure
End-stage heart failure due to ischaemic or dilated
cardiomyopathy is characterized by a dilated, thin-walled
ventricle. The structural basis for this ventricular dilatation is the side-to-side slippage of myocytes. However, probably of more importance is an increase in the
myocyte length secondary to an increase in the numbers
of sarcomeres in series [12,20]. The degree of side-to-side
slippage of myocytes does not correlate with the magnitude of ventricular enlargement ; furthermore, myocyte
lengthening alone may account for all the ventricular
dilatation seen in cardiomyopathy of various aetiologies
[12,20]. Similar changes of myocyte lengthening and
maladaptive remodelling of the cardiac myocyte begin in
hypertension long before ventricular failure occurs [20].
The mechanism for the initiation and maintenance of
increased circulating plasma CT-1 levels in patients with
heart failure is unknown. One possibility is that CT-1
expression may be induced by myocyte stretch. Indeed,
mechanical stretch activates the JAK\STAT pathway and
augments expression of CT-1 mRNA in rat cardiomyocytes [21]. It is intuitive to propose that the initial
secretion of CT-1 following ischaemic embarrassment
may be adaptive via : (i) cytoprotective effects potentially
conferred by this peptide ; (ii) improvement in cardiac
output and haemodynamics ; and (iii) series addition of
sarcomeres, resulting in a distribution of augmented wall
stress against an increased number of myofibrils. However, prolonged secretion of CT-1 may be deleterious,
resulting in excessive ventricular dilatation and irrevocable loss of function.
Our study has a number of important limitations. This
early report is limited by the relatively small number of
patients studied. We are currently investigating patterns
of secretion of CT-1 in larger numbers of patients with
chronic heart failure, following acute myocardial infarction and in unstable angina. The tendency to higher
levels of CT-1 in patients with clinical heart failure or in
those receiving diuretic or ACE inhibitor therapy may
simply reflect disease severity. However, the present
study does not address the issue of the response of CT-1
to pharmacological treatment of heart failure. Another
limitation of the present study is the inability to define a
direct cause-and-effect relationship between elevated
CT-1 levels and clinical parameters. Further to this, the
relationship between CT-1 and other cytokines known
to be elevated in heart failure remains to be addressed. In
this respect it is of interest to note that CT-1 stimulates
BNP gene expression and peptide secretion in cultured
rat myocytes [8,9]. This raises the intriguing possibility
that CT-1 may represent an earlier phase of the cascade of
neuroendocrine activation seen in heart failure. Thus
assessment of CT-1 and other neurohormones, such as
BNP, may provide differing but complementary information at different stages in the disease process.
Clearly additional studies with larger number of patients
are required.
The present study has identified a new signalling
pathway in human heart failure which could potentially
be manipulated therapeutically. Agonists or antagonists
of the CT-1 receptor may have therapeutic potential in
the protection of cardiac cells from ischaemic injury,
particularly if the cytoprotective properties can be
separated from the damaging hypertrophy-inducing effects that result from continuous activation of the gp130
signalling pathway. Additional clinical studies are
presently in progress, and may shed further light on the
role of this peptide in cardiovascular disease.
ACKNOWLEDGMENTS
We thank the British Heart foundation for its support.
S. T. was supported by a Leicester Royal Infirmary
research fellowship.
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Received 4 January 2000/9 March 2000; accepted 30 March 2000
# 2000 The Biochemical Society and the Medical Research Society