In the heart, Heat Shock Proteins (HSP) constitute an endogenous stress response that protect myocytes from damage. Although HSP are intracellular proteins, these can be found also in peripheral circulationwhere their increase is associated with early cardiovascular disease

Caselli, C

Del Ry, Silvia

Emdin, Michele

Khabirinejad, Jaleh

Neglia, Danilo

Passino, Claudio

PUblication MAnagement

Sunday 22 June 2003 57
313
Circulating antibodies to heat shock protein 65 in patients with
heart failure
D. Giannessi, C. Caselli, C. Passino, S. Del Ry, D. Neglia,
J. Khabirinejad, M. Emdin
CNR, Institute of Clinical Physiology, Pisa, Italy
Increased production of Heat Shock Proteins (HSP) is observed in the
cells, including cardiomyocytes, to protect themselves against stress stim-
uli. These proteins, that appear to be involved in repair processes and
have a number of cellular functions such as protein assembly and fold-
ing and transport crossing intracellular membranes, are also involved in
cardiovascular diseases. Increased circulating levels of Hsp 60 as well
as increased titers of antibodies against mycobacterial Hsp 65 have been
found to be associated with different cardiovascular diseases. Chronic
heart failure (CHF) is a situation of chronic stress, characterized by the
presence of factors, such as cytokines, able to increase the HSP. The
concentrations of HSP in the failing hearts are increased with respect to
nonfailing hearts, both in humans and in animal models. However data
about the presence of HSP or their antibodies in peripheral circulation in
this situation are so far lacking. For this, circulating anti-mycobacterial
65kDa protein antibodies were evaluated in 78 patients with CHF (ejec-
tion fraction, LVEF, 33.6 ± 0.87, 54 in I-II NYHA class and 24 in III-IV
class) and in 27 healthy subjects as control, by using an ELISA method.
Scalar dilutions (from 1:100 to 1:1600) of each serum sample were as-
sayed. Scalar dilution of a negative sample and of 2 different positive
samples were also evaluated in each run for quality control purpose. An-
tibodies to Hsp 65 were found in the 70% of the patients with CHF (titres
ranged from 1:100 to 1:1600) and in the 68% of controls (titres ranged
from 1:100 to 1:400). Mean (±SEM) antibody titres resulted: 222 ± 32
in controls, 304 ± 43 and 408 ± 55 in CHF patients in I-II and III-IV
NYHA class, respectively (p=0.035 III-IV class vs. controls by Fisher
test after ANOVA). No significant association between antibody titre and
degree of myocardial dysfunction, as assessed by LVEF, was found.
The positive correlation of the anti-Hsp 65 titres with clinical severity
(NYHA class) suggests a role for HSP in this disease, however the clinical
relevance of this determination has to be assessed by the comparison with
the distribution of anti-Hsp 65 antibodies in larger age- and sex-matched
healthy population (whose recruitment is now in progress), as well as by
the comparison with hemodynamic and biohumoral markers of CHF.
314
Heat shock protein 72 in heart failure
C. Caselli, C. Passino, S. Del Ry, D. Neglia, J. Khabirinejad, M. Emdin,
D. Giannessi
CNR, Institute of Clinical Physiology, Pisa, Italy
In the heart, Heat Shock Proteins (HSP) constitute an endogenous stress
response that protect myocytes from damage. Although HSP are intracel-
lular proteins, these can be found also in peripheral circulation where their
increase is associated with early cardiovascular disease. Among the HSP,
Hsp 72, the inducible isoform of the 70-kDa family, is considered as the
most responsive to a variety of stresses and experimental evidences indi-
cate that the Hsp 72 has an important function in cardioprotection. Heart
failure (CHF) represents a condition of chronic sustained stress charac-
terized by the overexpression of factors able to increase HSP, such as
cytokines. In humans, the heart expression of the main HSP is increased
in CHF with respect to nonfailing hearts. Since no data are available on
the circulating levels of Hsp 72 in patients with CHF, we deemed it in-
teresting to assess the serum levels of Hsp 72 in patients with diagnosis
of heart failure (CHF) and to investigate its association with the severity
of disease. Serum levels of Hsp 72 were measured in 127 patients with
CHF due to dilated cardiomyopathy (82 in I-II NYHA class and 45 in
III-IV class) by using an ELISA method (Stressgen, Canada)(sensitivity:
0.47 ± 0.21 ng/ml n=7, mean ± SD; between-assay variability: 10.8 ±
0.89 ng/ml, CV%: 8.2 and within-assay variability: 10.6 ± 0.41, ng/ml,
CV%:3.9).
Measurable amounts of circulating Hsp 72 were observed in the 90%
of the patients studied. The range of the Hsp 72 concentrations was 0.4
- 21.6 ng/ml. A tendency to increase of the serum levels of Hsp 72 as
a function of the NYHA class was observed (3.6 ± 0.43 ng/ml in I-II
NYHA class vs. 4.3 ± 0.46 in III-IV class, mean ± SEM, p=ns). A weak
negative relationship with the values of ejection fraction (LVEF%) was
also found.
To evaluate the involvement of this stress protein in the progression of
CHF, its association with functional parameters that reflect more accu-
rately the severity of disease than LVEF, as well as the comparison with
an age- and sex-matched control group, now in progress, are of pivotal
importance. Moreover, the possibility to reliably measuring HSP in the
peripheral circulation substantially increases the possibility of studying
the role of these proteins in various pathophysiological processes.
315
P19 embryonal carcinoma cells: a suitable model system for cardiac
electrophysiological differentiation at the molecular and functional
level
M.A.G. Van der Heyden1, M.J. Van Kempen1, Y. Tsuji2, M.B. Rook1,
H.J. Jongsma1, T. Opthof1
1University Medical Centre Utrecht, Medical Physiology, Utrecht,
Netherlands; 2RIEM, University of Nagoya, Dept of Circulation,
Nagoya, Japan
Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate
into spontaneously beating cardiomyocytes in vitro and have revealed
important insight into the early molecular processes of cardiomyocyte
differentiation. We assessed the suitability of the P19 cell model for
studying cardiac ion channel regulation at the molecular and functional
level.
Methods: P19 cells were induced to differentiate towards cardiomy-
ocytes. mRNA’s for cardiac markers and ion channels were determined
by RT-PCR at 6 timepoints during the differentiation process. Action po-
tentials and individual ion currents were measured by whole cell patch
clamp.
Results: Ion channel mRNA expression of several channels is temporally
regulated during differentiation, while others show little or no regulation.
L-type calcium and transient outward channels are expressed from very
early on, while sodium and delayed and inward rectifier channels are up-
regulated at somewhat later stages during differentiation, which mirrors
the in vivo murine cardiomyocyte differentiation during embryogenesis.
Spontaneous cardiomyocyte action potentials exhibit a low upstroke ve-
locity, which often can be enhanced by hyperpolarizing the cells, hence
activating thusfar dormant ion channels to contribute to the action poten-
tial upstroke. Action potential duration decreases considerably during the
differentiation of spontaneously beating cells. In late stages, non-beating
myocytes can be found which only generate action potentials upon elec-
trical stimulation. Their shape is comparable to neonatal/juvenile ven-
tricular mouse myocytes in culture. Finally, we show that P19-derived
cardiomyocytes display a very complete set of functional ion channels.
Conclusion: P19 cells represent a powerful model to study the regulation
of myocardial electrophysiological differentiation at the molecular and
functional level.
316
Role of calcineurin in chronic hypoxia-induced hypertrophic
response of right ventricle
N. Koulmann1, H. Sanchez1, A. Letout1, R. Ventura-Clapier2,
X. Bigard1
1CRSSA, Département des facteurs humains, La Tronche, France;
2INSERM U-446, Laboratoire de Cardiologie moléculaire,
Chatenay-Malabry, France
Chronic exposure to hypoxia leads to pulmonary hypertension and then
to a significant right ventricle (RV) hypertrophy. It is well known that
this hypertrophic process is associated with an increased proportion of


Heat shock protein 72 in heart failure

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Heat shock protein 72 in heart failure

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