Reduced cardiac hypertrophy and altered blood pressure control in transgenic rats with the human tissue kallikrein gene

Reduced cardiac hypertrophy and altered blood
pressure control in transgenic rats with the human
tissue kallikrein gene1
JOSE
´ -A. SILVA, JR.,*,† RONALDO C. ARAUJO,*,† OVIDIU BALTATU,*
SUZANA M. OLIVEIRA,*,† CARSTEN TSCHO
¨ PE,‡ EDWIN FINK,§ SIGRID HOFFMANN,*
RALPH PLEHM,* KARL X. CHAI,¶ LEE CHAO,¶ JULIE CHAO,¶ DETLEV GANTEN*,††
JOA
˜ O B. PESQUERO1,2 AND MICHAEL BADER*,2
*Max-Delbru
¨ ck-Center for Molecular Medicine, Berlin-Buch, Germany; †Department of Biophysics,
Escola Paulista de Medicina, Sa˜o Paulo, Brazil; ‡Departments of Cardiology and ††Clinical
Pharmacology, University Hospital Benjamin Franklin, Free University, Berlin, Germany;
§Department of Clinical Chemistry and Clinical Biochemistry, University of Munich, Germany; and
¶Department of Biochemistry and Molecular Biology, Medical University of South Carolina,
Charleston, South Carolina 29425, USA
SPECIFIC AIM
24 h period in TGR(hKLK1): 1.6?0.2; in SD:
2.7?0.4; P?0.05). However, in both strains, the
The aim of this study was to evaluate the cardio-
acrophases of the 24 h period occurred around
vascular actions of kinins, which have been impli-
midnight for BP, and the rhythms of heart rate and
cated in the bene?cial effects of angiotensin-convert-
locomotor activity were similar.
ing enzyme (ACE) inhibitors by the generation and
analysis of a transgenic rat line harboring the human
3. Reduced cardiac hypertrophy and ?brosis in
tissue kallikrein gene, TGR(hKLK1).
TGR(hKLK1)
To study the role of kallikrein in cardiac hypertro-
PRINCIPAL FINDINGS
phy and ?brosis, TGR(hKLK1) and SD control rats
were treated with a suppressor dose of isoproterenol
1. Transgene expression
for 7 days. This treatment resulted in a marked
increase in relative heart (Fig. 2A) and left ventricu-
Expression of a human tissue kallikrein transgene
lar weight (Fig. 2B) in both strains of rats. However,
(hKLK1) under the control of the mouse metallo-
the effect was signi?cantly less pronounced in
thionein promoter was detected in all organs of the
TGR(hKLK1), indicating a protective action of trans-
newly generated transgenic rat line, TGR(hKLK1)
gene expression. Expression of ANP in the left
(Fig. 1). Translation of the hKLK1-mRNA was veri-
ventricle, an early marker of cardiac hypertrophy,
?ed by the demonstration of human kallikrein in the
supported these ?ndings. Whereas it was markedly
urine of transgenic rats (700 ? 127 ng/ml).
induced in SD rats, no effect on ANP expression was
observed in TGR(hKLK1) (Fig. 2C). Kinins and their
2. Blood pressure
B2 receptors obviously mediated the kallikrein ac-
tion as the transgene effect on cardiac hypertrophy
Mean arterial pressure determined by telemetric
was abolished by the coadministration of icatibant
measurement turned out to be slightly but signi?-
(Fig. 2).
cantly lower in TGR(hKLK1) animals compared to
Moreover, interstitial ?brosis in the left ventricle,
Sprague Dawley (SD) control rats (110.5?1.1 vs.
also induced by isoproterenol treatment and quan-
114.9?1.0 mmHg; P?0.01). In contrast, no signi?-
ti?ed by detecting collagen III mRNA in left ventri-
cant difference was observed between the transgenic
cles, was less pronounced in TGR(hKLK1) com-
and SD rats with respect to heart rate or locomotor
pared to SD rats (Fig. 2D).
activity. The B2 antagonist icatibant increased blood
pressure (BP) signi?cantly by 2.0 ? 0.9 mmHg
1
(P?0.01) only in TGR(hKLK1).
To read the full text of this article, go to http://www.
fasebj.org/cgi/doi/10.1096/fj.99-1010fje
The 24 h rhythm of mean arterial pressure in
2 Correspondence: Max-Delbru¨ck-Center for Molecular
TGR(hKLK1) animals was dampened in comparison
Medicine (MDC), Robert-Ro¨ssle-Strasse 10, D-13092 Berlin-
to untreated SD rats (amplitude of the dominant
Buch, Germany. E-mail: mbader@mdc-berlin.de
1858
0892-6638/00/0014-1858/$02.25 © FASEB

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Figure 1. Ribonuclease protection assay
detects expression of the hKLK1 gene in
different organs of TGR(hKLK1) (?) but
not in SD control rats (?). 20 ?g of total
RNA from tissues of individual animals or
yeast (Y) together with hKLK1- and ?-actin-
speci?c probes was used.
CONCLUSIONS AND SIGNIFICANCE
role of the kallikrein-kinin system (KKS) in circadian
?uctuations of arterial pressure. The biological
Transgenic rats expressing human tissue kallikrein
mechanisms underlying regulation of circadian
at high levels in all organs investigated became
rhythms of cardiovascular parameters are largely
hypotensive probably through increased generation
unknown. In mammals, most circadian rhythms are
of kinins as evidenced by a partial normalization of
governed by what appears to be the main internal
BP after treatment with the B2 receptor antagonist
oscillator, the suprachiasmatic nucleus of the hypo-
icatibant. These ?ndings are in line with results
thalamus (SCN), since lesions in this brain region
obtained in mice expressing the same transgene and
can obliterate the rhythms of heart rate and BP.
hypertensive rats after somatic gene transfer of the
Nonetheless, little is known about how oscillations in
hKLK1 gene.
the SCN are biochemically achieved or which neu-
An important novel observation in this study is the
ronal or hormonal pathways are used to establish the
Figure 2. Cardiac hypertrophy and ?brosis after isoproterenol treatment is reduced in TGR(hKLK1) compared to SD rats. Heart
weight (A) and left ventricular weight (B) to body weight ratios were determined of untreated SD and TGR(hKLK1) rats and
of animals treated with isoproterenol (ISO) alone for 7 days (n?10) or with coadministration of icatibant (HOE, n?4). ANP
(marker of hypertrophy, C) and collagen III (marker of ?brosis, D) expression were detected by ribonuclease protection assay
in left ventricles of untreated and ISO-treated SD and TGR(hKLK1) rats (upper panel). 20 ?g of total RNA of left ventricles was
used with probes for rat ANP, collagen III, and ?-actin. The levels of ANP and collagen III mRNA relative to ?-actin mRNA were
quanti?ed using the software TINA on a PhosphorImager system (lower panel). ***P ? 0.005, **P ? 0.01 vs untreated rats; ###P
? 0.005, ##P ? 0.01, #P ? 0.05.
HUMAN TISSUE KALLIKREIN TRANSGENIC RATS
1859

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ators of the transgene effects. Consistent with an
important role of kinins, previous pharmacological
studies have shown that isoproterenol-induced car-
diac hypertrophy can be diminished more effectively
by ACE inhibitors than by AT1 antagonists. Kinins
have also been implicated in the antihypertrophic
actions of ACE inhibitors in the aortic coarctation
model since icatibant effectively blunted the drug-
induced reduction of left ventricular hypertrophy.
Furthermore, bradykinin infusion prevented devel-
opment of increased cardiac mass in the same
model. While kinins may act as weak growth factors
on cultured cardiomyocytes and ?broblasts, they
have also been shown to reduce cardiac collagen
synthesis and growth via release of prostaglandins
and nitric oxide. According to our results, the latter
seems to be the prevailing in vivo effect. Adaptation
Scheme 1
of cardiac muscle to an increased work load can be
achieved either by an increase in muscular mass (i.e.,
rhythm of the cardiovascular system. Kallikrein, kini-
hypertrophy) or by an improved performance of
nogens, kinins, and their receptors, B1 and B2, are
the existing myocardium. Kinins may inhibit hyper-
present in several regions of the brain, including
trophy by improving the supply of cardiomyocytes
SCN, and the central KKS is involved in BP regula-
with nutrients and oxygen via two independent
tion. Furthermore, a circadian rhythm of kallikrein
mechanisms: increase in coronary perfusion and
expression has recently been reported in the rat
stimulation of myocardial glucose uptake. These
pineal gland. Kinins may also be important in trans-
kinin effects may be crucial for the prominent car-
mitting the oscillations generated in SCN and pineal
dioprotective actions of ACE inhibitors. In summary,
gland to other brain areas or to the periphery. A
we have developed a new transgenic rat model with
peripheral action of the KKS governing diurnal BP
an overactive KKS. These rats may be applicable to
variations is supported by the circadian rhythm of
the study of multiple issues of cardiovascular regula-
urinary kallikrein excretion reported, with highest
tion and other physiological and pathophysiological
concentrations preceding lowest BP values. Thus,
mechanisms in which kinins might participate.
the KKS may be deeply involved in the circadian
regulation of BP. As the circadian rhythmicity of
This work was supported by grants from the European
heart rate is unaltered in TGR(hKLK1), this study
Union (Transgeneur), the Deutscher Akademischer Aus-
con?rms earlier reports that the rhythms of BP and
tauschdienst (PROBRAL), the Fundac¸a˜o de Amparo a` Pas-
heart rate are differentially regulated.
quisa do Estado de Sa˜o Paulo (FAPESP), and the Volkswa-
The most important novel ?nding of this study is
genstiftung. R.C.A. and J.B.P. received fellowships from the
Max-Delbru
¨ ck Center for Molecular Medicine and J.A.S. from
the prohibitive effect of tissue kallikrein in isoprot-
FAPESP. We thank Dr. K. Wirth (Hoechst Marion Roussel)
erenol-induced cardiac hypertrophy and ?brosis. Re-
for his generous gift of icatibant and Dr. X. Tian for the
ductions in hypertrophy and ?brosis were abolished
collagen III probe. The excellent technical assistance of M.
by icatibant, which further supports kinins as medi-
Somnitz and A. Bo¨ttger is gratefully acknowledged.
1860
Vol. 14 October 2000
The FASEB Journal
SILVA ET AL.

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