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Cardiac Thyrotropin-Releasing Hormone Mediates Left
Ventricular Hypertrophy in Spontaneously
Hypertensive Rats
Mariano L. Schuman, Maria S. Landa, Jorge E. Toblli, Ludmila S. Peres Diaz, Azucena L. Alvarez,
Samuel Finkielman, Leonardo Paz, Gabriel Cao, Carlos J. Pirola, Silvia I. García
See Editorial Commentary, pp 26 –28
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Abstract—Local thyrotropin-releasing hormone (TRH) may be involved in cardiac pathophysiology, but its role in left
ventricular hypertrophy (LVH) is still unknown. We studied whether local TRH is involved in LVH of spontaneously
hypertensive rats (SHR) by investigating TRH expression and its long-term inhibition by interference RNA
(TRH-iRNA) during LVH development at 2 stages (prehypertrophy and hypertrophy). SHR and their control rats
(WKY) were compared. Cardiac hypertrophy was expressed as heart/total body weight (HW/BW) ratio. TRH content
(radioimmuno assay), preproTRH, TRH receptor type I, brain natriuretic peptide (BNP), and collagen mRNA
expressions (real-time polymerase chain reaction) were measured. For long-term inhibition of TRH, TRH-iRNA was
injected into the left ventricle (LV) wall for 8 weeks. Hearts were processed for morphometric studies and
immunohistochemical analysis using antibodies against ␣-smooth muscle actin and collagen type III. LV preproTRHmRNA abundance was similar in both strains at 7 weeks of age. At the hypertrophic stage (18 weeks old), however,
there was a 15-fold increase in SHR versus WKY, consistent with a significant increase in tripeptide levels and the
expression of its receptor. Specific LV-TRH inhibition at the prehypertensive stage with TRH-iRNA, which decreased
⬎50% preproTRH expression and tripeptide levels, prevented LVH development as shown by the normal HW/BW ratio
observed in TRH-iRNA–treated SHR. In addition, TRH-iRNA impeded the increase in BNP and type III collagen
expressions and prevented the increase in cardiomyocyte diameter evident in mismatch iRNA-treated adult SHR. These
results show for the first time that the cardiac TRH system is involved in the development of LVH in SHR. (Hypertension.
2011;57:103-109.) ● Online Data Supplement
Key Words: TRH 䡲 cardiac hypertrophy 䡲 rat 䡲 SHR 䡲 interference RNA
T
not be regulated by T3.8 In addition, Hasegawa et al9 reported
for the first time an inotropic effect of TRH on the guinea pig
myocardium, implying that this effect could be mediated by
an increase in a slow inward Ca2⫹ current. Furthermore,
Socci et al7 found similar results and reported that TRH
modulates cardiac contractility of isolated rat hearts as an
autocrine factor in a concentration-dependent manner, involving more than one TRH receptor. Enhanced ventricular
contractility by TRH was also demonstrated by other authors
in both open-chested dog preparation and ex vivo ventricular
myocytes, using video edge cinematography.8
More recently, using DNA microarrays, Jin et al10 identified preproTRH as one of the genes induced in the left
ventricle (LV) of rats with heart failure, 8 weeks after
hyrotropin-releasing hormone (TRH), a small neuropeptide (p-Glu-His-Pro-NH2) initially identified in the hypothalamus, is amply distributed in the central nervous
system1 and in other extraneural tissues2 and has been shown
to have central and peripheral biological effects independent
of thyroid hormone production.3 TRH also acts on the
cardiovascular system of rodents.4,5
Many groups have identified preproTRH-mRNA by Northern blot analysis and RNAse protection assay in rat cardiac
tissues and have referred the presence of specific type I TRH
receptors (TRH-R1) in ventricles, establishing that a TRH
system is present in the rat heart.6 – 8 In contrast to the
hypothalamic TRH system, cardiac preproTRH-mRNA may
be augmented by glucocorticoids and by testosterone but may
Received August 13, 2010; first decision August 28, 2010; revision accepted October 29, 2010.
From the Departamento de Cardiología Molecular (M.L.S., M.S.L., L.S.P.D., S.F., S.I.G.), Departamento de Genética y Biología Molecular de
Enfermedades Complejas (M.S.L., A.L.A., C.J.P.) and Servicio de Anatomía Patológica (L.P.), Instituto de Investigaciones Médicas Alfredo Lanari,
Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Investigaciones Medicas and Consejo Nacional de Investigaciones Cientificas y
Tecnicas (IDIM-CONICET) (J.E.T., G.C.), Laboratory of Experimental Medicine, Hospital Alemán, Buenos Aires, Argentina.
M.S.L., J.E.T., S.F., C.J.P., and S.I.G. belong to the National Council of Scientific and Technological Research.
Correspondence to Silvia I. García, Departamento de Cardiología Molecular, Instituto de Investigaciones Médicas Alfredo. Lanari, UBA,
IDIM-CONICET, Combatiente de Malvinas 3150, 1427 Buenos Aires, Argentina. E-mail [email protected]
© 2010 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.110.161265
103
January 2011
myocardial infarction. They also suggested that TRH can
increase cardiac performance in rats with ischemic cardiomyopathy.11 Based on these results, TRH was postulated to play
an autocrine or paracrine role in cardiac physiology, although
the physiological role of endogenous TRH in the heart
remains unknown. Moreover, to our knowledge, no evidence
has been reported on the role of the cardiac TRH system on
cardiac hypertrophy.
In this study, spontaneously hypertensive rats (SHR) were
used as a cardiac hypertrophy model to investigate the role of
the LV cardiac TRH system in the development of left
ventricular hypertrophy (LVH).
We show, for the first time, the expression of TRH system
components in prehypertrophic and hypertrophied LVs of
SHR and developed a long-term inhibition of the LV TRH
system to evaluate the participation of the local TRH system
in the development of LVH.
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Methods
All reagents were from Sigma unless indicated. The Institutional
Animal Care and Use Committee approved the animal experimentation protocols following the ethical guidelines.
A
200
WKY
SHR
175
SBP (mmHg)
Hypertension
150
*
125
100
75
50
0
7 weeks
Male SHR and WKY rats (Charles River Laboratories, Wilmington,
MA) (10 per group) at 7 weeks old and at the hypertensive stages (18
weeks old) were used. The animals were housed in a room with
controlled temperature (23⫾1°C) under a 12-hour light/dark schedule. Before the experiments, blood pressure was recorded.
In Vivo Interference RNA Treatment
Seven-week– old SHR were assigned in a random blind fashion to 1
of the 2 groups, (1) specific TRH-interference RNA (TRH-iRNA)
and (2) scramble mismatched control (Con-iRNA) (n⫽10 per
group), and were anesthetized (90 mg/kg ketamine and 10 mg/kg
xylazine). Injections were conducted every 2 weeks during the
experiment (8 weeks) to maintain the knockdown effect. All injections were done under echography.
For a detailed Materials and Methods, please see the online Data
Supplement, available at http://hyper.ahajournals.org.
Results
LV-TRH Hyperactivity in the
Hypertrophied Ventricle
We used SHR at 2 different ages and their controls, agematched WKY. At the age of 7 weeks the SHR presented a
small increase in blood pressure without any difference in the
heart/total body weight (HW/BW) ratio with respect to the
normotensive strain, in comparison with adult SHR (18
weeks old), which were hypertensive and showed a markedly
hypertrophied heart with a significant increase in the
HW/BW ratio (Figure 1). As we had hypothesized, we found
an age-dependent and significant increase in TRH expression
in the LV of SHR with respect to the normotensive and
normotrophic WKY rats, as TRH content increased 6-fold at
18 weeks when LVH was marked (Figure 2A). In contrast, at
the prehypertrophic stage, no significant changes between
both strains were observed. As changes in peptide content
could reflect both differential posttranslational processing
and expression, we also evaluated preproTRH-mRNA abundance. The increase in TRH content at the hypertrophic stage
was accompanied by a significant increase in the preproTRH-
18 weeks
†
B
0.40
WKY
SHR
*
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
7 weeks
Animals
†
*
25
Heart weight / Body weight
g/g x 100
104
18 weeks
Figure 1. Systolic blood pressure (SBP; A) and HW/BW ratio (B)
of prehypertensive (7 weeks old) vs hypertensive (18 weeks old)
SHR compared to their age-matched control WKY rats (n⫽10
per group). *P⬍0.05 SHR vs WKY; †P⬍0.05 between different
ages from the same strain.
mRNA abundance in the SHR LV (Figure 2B) compared to
their age-matched WKY controls (P⬍0.01). In parallel with
these results, we also found a significant increase in the
specific type 1 receptor (TRH-R1) gene expression compared
to the 7-week– old SHR; a smaller increase was also seen
when compared to the age-matched WKY rats, although it did
not reach statistical significance. Peptide, precursor, and
specific receptor results showed a hyperactivity of the TRH
system in the hypertrophied ventricle. No significant changes
were seen in the WKY strain at both stages.
SHR LV changes seem to be specific since no changes
were found in the right ventricle at this age either in TRH
content (18 weeks WKY 0.6⫾0.1 pg/mg protein, 18 weeks
SHR 0.55⫾0.2 pg/mg protein) or preproTRH-mRNA abundance (18-week WKY 5.7⫾0.5 arbitrary units, 18-week SHR
6.55⫾0.5 arbitrary units). We observed a slight but not
significant increase in TRH content in the septum of adult
SHR (18-week WKY 0.35⫾0.1 pg/mg protein, 18-week SHR
0.47⫾0.15 pg/mg protein) that was, accordingly, similar to
the septum preproTRH-mRNA abundance found between
both strains (18-week WKY 6.3⫾0.35 arbitrary units, 18week SHR 5.85⫾0.42 arbitrary units).
These results pointed out an activation of the TRH system
on the hypertrophied LV of SHR.
LV-TRH Long-Term Inhibition by Intracardiac
iRNA Treatment
First, we evaluated the efficacy of the specific TRH-iRNA
treatment, and we measured preproTRH-mRNA abundance
and TRH content in the LV of the SHR injected with the
specific TRH-iRNA or the mismatched Con-iRNA oligonu-
Schuman et al
A
3.5
Left Ventricle
TRH pg/mg protein
3.0
†
WKY
SHR
‡
7 weeks
18 weeks
2.5
2.0
1.5
1.0
0.5
0.0
Left Ventricle TRH-R1
C
10
8
6
4
2
WKY
SHR
†
‡
0.1
0.0
expression TRH − R1/ β -actin
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Left Ventricular preproTRH
expression preproTRH / β -actin
B
7 weeks
14
18 weeks
WKY
SHR
12
†
10
8
6
4
2
0
7 weeks
18 weeks
Figure 2. Left ventricular TRH overexpression in adult SHR. LV
TRH content (picogram TRH/milligram protein) measured by
radioimmuno assay (A), TRH precursor (preproTRH; B), and TRH
receptor type 1 (TRH-R1; C) expressions determined by realtime polymerase chain reaction normalized by ␤-actin expression in SHR vs control WKY rats at the prehypertrophic (7
weeks old) compared to the hypertrophic (18 weeks old) stage
(n⫽10 per group). ‡P⬍0.01 SHR vs WKY; †P⬍0.01 between
different ages from the same strain.
cleotides. As shown in Figure 3A (left), the inhibition of TRH
by specific iRNA treatment (SHR⫹TRH-iRNA) decreased
LV preproTRH gene expression by 53% compared to the
same strain treated with control oligos (SHR⫹Con-iRNA).
A tendency of decrease was observed in the septum of the
TRH-iRNA group, but it did not reach statistical significance
(Figure 3A, right). These results were also confirmed by the
TRH content measured by radioimmuno assay. No significant
changes in preproTRH-mRNA abundance were seen either in
atria or right ventricle.
As shown in Figure 3B, the specific and long-lasting
inhibition of LV-TRH expression impeded the expected
increase of the hypertrophy index in adult SHR (18 weeks
old). Moreover, the TRH-iRNA–treated SHR presented a
HW/BW ratio similar to that of control normotensive WKY
rats, indicating the normotrophic stage of the treated SHR
(Figure 3B, left). These results were confirmed using the
TRH and Cardiac Hypertrophy in SHR
105
HW/snout-tail long index; again (Figure 3B, right), WKY and
TRH-iRNA–treated SHR showed similar indices that were
significantly lower compared to those of the Con-iRNA–
treated SHR.
These normal ratios found in the animals with the inhibition of the LV TRH were not caused by normalization of
blood pressure or changes in the body weight, as we measured both variables weekly during the entire experiment, and
we did not find any significant changes. (Figure 3C).
In addition, we characterized the effect of the long-lasting
inhibition of LV TRH on molecular expression of genes
previously shown to be involved in the progression and
widely recognized as markers of cardiac hypertrophy, such as
brain natriuretic peptide (BNP) and another involved in
fibrosis development, the type III collagen, in both groups of
hypertensive treated rats. As observed in Figure 4A, the
group of SHR with LV-TRH inhibition showed a 55%
decrease in type III collagen expression compared to control
mismatched iRNA-treated SHR. In accordance, a significant
reduction in BNP expression was observed in the same group
compared to control mismatch-iRNA–treated SHR. Together,
these results suggest that the specific TRH inhibition in the
LV may be protective for the heart.
To confirm the specificity of the in vivo iRNA treatment,
we decided to quantify preproTRH gene expression on the
diencephalum, because it is known that the TRH system
participates not only in the endocrine system but also in
cardiovascular regulation12 and in other extraneuronal tissues
where the tripeptide has been found.13 We also evaluated the
thyroid status in both groups of animals. As shown in Figure
4B, cardiac iRNA treatment against TRH did not alter either
the diencephalic preproTRH gene expression (right) or
plasma thyroid hormone levels (left) in any group. In addition, no differences between the 2 groups were seen in
pancreas and liver (data not shown), indicating that the
treatment was tissue specific and its effects were independent
of the thyroid system.
To further confirm the reversion of LVH by TRH
inhibition, histological studies of LV in both groups were
performed. The specific TRH-iRNA treatment prevents the
significant increase in the cardiomyocyte diameter, which
was expected and evident only in the SHR treated with the
scrambled control iRNA, as shown in the supplemental
table.
According to the cardiomyocyte diameter, TRH-iRNAtreated SHR present a significant increase in myocyte
density, evaluated as the number of cells per area and as
the number of capillary per area, indicating that LV-TRH
inhibition prevents hypertrophy development in SHR. In
line with these findings, similar changes in the interventricular septum were observed in the SHR⫹TRH-iRNA
group, suggesting that the TRH-iRNA reach the entire LV,
as was expected.
As illustrated in Figure 5, evaluation of extracellular matrix
(ECM) expansion by Masson’s trichrome (A, B) and Sirius
red (C, D) stains showed that LV-TRH inhibition prevents
(P⬍0.01) the increase in ECM expected for an adult SHR.
Furthermore, LV-TRH inhibition prevents the increase of
␣-smooth muscle actin, a protein related to the fibrosis
Hypertension
90
60
#
30
0
SHR +
TRH-iRNA
SHR +
Con-iRNA
*
0.0040
0.0035
#
0.0030
0.0025
0.0020
0.0015
125
100
75
50
25
0
SHR +
TRH-iRNA
SHR +
Con-iRNA
0.100
*
0.075
#
0.050
0.025
0.000
WKY
C
SHR +
Con-iRNA
WKY
SHR +
TRH-iRNA
SHR + Con-iRNA
SHR + TRH-iRNA
440
SHR +
Con-iRNA
SHR +
TRH-iRNA
SHR + Con-iRNA
SHR + TRH-iRNA
200
400
SBP (mmHg)
Body weight (g)
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Heart weight/Body weight
(g/g)
B
120
Septum
PreproTRH expression
TRH/β -actin ( % of control)
Left ventricle
PreproTRH expression
TRH/β-actin ( % of control)
A
January 2011
Heart weight/Snout-tail long
(g/cm)
106
360
320
180
160
140
280
240
7.0
9.5
12.0
14.5
17.0
Age (weeks)
120
7.0
9.5
12.0
14.5
17.0
Age (weeks)
Figure 3. Evidence of the mediator role of TRH in SHR-cardiac hypertrophy. A, Evaluation of the specific TRH-iRNA treatment on
preproTRH-mRNA precursor expression in LV and septum. B, TRH-iRNA prevented cardiac hypertrophy in SHR as shown by 2 ratios,
HW/BW (left) and heart weight/snout-tail long (right). C, Body weight (left) and systolic blood pressure (SBP) (right) in SHR treated with
Con-iRNA or specific TRH-iRNA during the experiment (8 weeks); arrows indicate intracardiac injections. *P⬍0.05 compared with the
WKY control group; #P⬍0.05 compared with the SHR⫹Con-iRNA group; n⫽10.
process and expressed in interstitial myofibroblasts as shown
in Figure 5E and 5F.
In favor of a better conserved cardiac structure in the
LV-TRH–inhibited group and in agreement with the observation of 55% less type III collagen mRNA expression, a
substantial reduction (P⬍0.01) in collagen III immunostaining in the LV-TRH–inhibited SHR group with respect to the
control SHR was observed (Figure 5G and 5H).
Finally, Figure 5I shows bars representing the quantification of ECM expansion, ␣-smooth muscle actin, and collagen
III immunostaining areas in both groups of animals.
Discussion
Many groups have defined SHR as a good model for human
cardiac pathophysiology, in particular LVH.14 Since 1992, when
a cardiac TRH system was reported, many authors have been
studying the role of local TRH in cardiac physiology.6 – 8,15 An
increase in TRH expression in the ventricle of infarct rats
after an 8-week injury has been reported, suggesting the
participation of the tripeptide in the cardiac damage.11 Thus,
the aim of this study was to evaluate the role of the cardiac
TRH system in the LVH of SHR. To further characterize the
changes in the TRH system during cardiac hypertrophy
development, we examined SHR at 2 stages, at 7 and at 18
weeks of age, to encompass the heart shift from normotrophic
to hypertrophic phenotype. As expected, no changes either in
TRH content or in preproTRH-mRNA abundance were found
at 7 weeks when both hypertension and hypertrophy were
absent, as seen by comparing normotensive with respect to
hypertensive strain, showing that alterations attributed only to
genetic differences between strains were unlikely.
Interestingly, in accordance with our hypothesis, we found
a marked increase in the expression of the cardiac TRH
system (preproTRH-mRNA, peptide, and its specific receptor) in the hypertrophied LV, as confirmed by the significant
increase in BNP expression and the hypertrophy indices used.
Schuman et al
125
100
75
#
50
25
0
SHR +
Con-iRNA
B
Type III collagen expression
III collagen/β -actin
(% of control)
BNP expression
BNP/ β -actin (% of control)
A
SHR +
TRH-iRNA
107
125
100
75
#
50
25
0
1.5
5
1.0
3
2
0.5
1
0
0.0
µ dl)
Serum T3 (µg/
4
Diencephalic preproTRH expression
TRH/ β-actin (% of control)
6
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SHR +
Con-iRNA
SHR +
TRH-iRNA
SHR +
Con-iRNA
SHR +
TRH-iRNA
SHR + Con-iRNA
SHR + TRH-iRNA
Serum T4 (ng/ml)
TRH and Cardiac Hypertrophy in SHR
125
100
75
50
25
0
Figure 4. Effect of long-term left ventricular TRH inhibition on hypertrophy markers expression and thyroid status in adult SHR treated
with the specific TRH-iRNA compared with the control treatment. A, BNP and type III collagen mRNA were determined by real-time
polymerase chain reaction and normalized by ␤-actin expression. Results are expressed as percentage of control group. B, Plasma
thyroid hormones levels, tyroxine (T4, nanograms per milliliter) and tryiodine (T3, micrograms per deciliter) (Roche-ECLIA), and diencephalic TRH precursor (diencephalic preproTRH) expression determined by real-time polymerase chain reaction and normalized by
␤-actin expression in adult SHR treated with the specific TRH-iRNA (SHR⫹TRH-iRNA) compared to control-iRNA treatment
(SHR⫹Con-iRNA). #P⬍0.05 compared with the SHR control group (SHR⫹Con-iRNA); n⫽10.
These data suggest the possible participation of the cardiac
LV TRH in this pathology either as a promoting or a protective
factor.
The association of cardiac TRH with hypertrophy development is supported by our findings that TRH peptide, its
precursor, and its specific receptor are upregulated only on
the hypertrophied LV as no changes were seen in other
cardiac tissue (right ventricle or septum).
To our knowledge, this is the first description of hyperactivity of the cardiac TRH system in the hypertrophied LV of
adult SHR, indicating that this hyperactivity is associated
with the development of the hypertrophy.
As there is no commercial TRH antagonist available, to
further investigate the TRH role in this model, we developed
a long-term inhibition of the TRH system in the LV of adult
SHR by using iRNA against the preproTRH gene and
demonstrated that the reduction of the augmented LV-TRH
expression in SHR impedes LVH development, showing for
the first time that LV TRH promotes hypertrophy in this
model.
In fact, specific TRH-iRNA treatment that normalizes the
augmented TRH-LV expression prevents the increase of the 2
hypertrophy ratios expected in adult SHR. Moreover, no
differences were seen between the normal WKY rats and the
SHR treated with the specific TRH-iRNA, indicating that the
treatment was efficient and avoids detrimental TRH actions
on cardiac tissue. This reduction in the indices were not
caused by differences in body weight as the animals did not
show any body weight difference along the treatment between groups. Despite the fact that systolic blood pressure
levels remained high in both groups of hypertensive rats,
TRH-iRNA normalized LVH even when overload was maintained. These results suggest that the group in which hypertrophy development was avoided would develop a more
severe cardiac failure, as it has been described that in a first
phase, LV hypertrophy helps the heart to function with a
higher requirement. However, additional investigations will
be necessary as we did not follow the protocol for more than
8 weeks.
In accordance with pathology results, we were able to
observe that LV-TRH inhibition impedes not only the
increase of the hypertrophy marker gene expression BNP
but also collagen III expression and content, indicating that
hypertrophy and fibrosis development was impeded at the
level characterized by these sensitive markers despite the
high blood pressure.
The normal levels of BNP expression in the LV of
TRH-iRNA–treated SHR was accompanied by a reduced
atrial natriuretic peptide expression (data not shown), indicating a regression of the hypertrophied tissue to the fetal
gene expression program. Finally, pathology studies were
able to confirm the benefit of the specific TRH-iRNA
108
Hypertension
January 2011
SHR +
TRH-iRNA
SHR +
Con-iRNA
A
B
SHR +
TRH-iRNA
SHR +
Con-iRNA
C
100 µm
SHR +
TRH-iRNA
SHR +
Con-iRNA
100 µm
F
100 µm
SHR +
TRH-iRNA
G
percentage / area (mm2 )
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E
I
100 µm
D
100 µm
24
22
20
18
16
14
12
10
8
6
4
2
0
SHR +
Con-iRNA
Perspectives
H
100 µm
SHR + TRH-iRNA
SHR + Con-iRNA
* p< 0.01, n=5
*
Additionally, adult SHR treated with the specific TRHiRNA showed absence of fibrosis in compared to the SHR
treated with mismatch-iRNA, as we observed with the immunostaining for collagen III and Sirius red techniques.
iRNA has been shown to be efficient for specific gene
silencing16; indeed, potential effects on other tissues may not
be significant, as we were able to show that effects were
independent of central TRH and thyroid status.
Although this in vivo intracardiac treatment, which involved repeated injections of naked iRNA, is not feasible as
therapy, these results show a useful tool to identify novel
gene functions particular in the heart in rodents and also
suggest that intracardiac iRNA injections may be suitable for
long-term treatment. In fact, we demonstrate for the first time
that repeated intracardiac injections of a specific iRNA could
reduce the expression of the target gene.
The results in the present study demonstrate the following:
(1) there was TRH hyperactivity in the hypertrophied LV of
the SHR, and (2) an in vivo specific TRH-iRNA treatment
induced downregulation of LV-TRH production, preventing
cardiac hypertrophy and fibrosis development.
These findings suggest new potential approaches for heart
disease, as we demonstrated that TRH is involved in cardiac
hypertrophy and is associated with the hypertrophic and
fibrotic process, effects that can be disrupted by targeting
TRH.
*
*
Our results indicated that the tripeptide may induce heart
damage, where hypertrophy, fibrosis, and apoptosis could be
involved. Interestingly, an increased cell death occurs before
ABP increase in the SHR heart, as reported by Liu et al.17
Additional studies using cardiac cell cultures, as we found
specific type I TRH receptor expression in cultured neonatal
cardiomyocytes and fibroblasts (unpublished results), would
be necessary to elucidate the mechanism by which TRH
induces the development of LVH.
Sources of Funding
ECM
α-smooth muscle actin
Collagen III
Figure 5. Effect of long-term LV-TRH inhibition on ECM expansion, collagen III, and cardiomyocyte morphometry (⫻400). A, B,
Note the significant difference in ECM expansion (arrows)
between groups (Masson’s trichrome). C, D, Arrows indicate
areas with a different degree of fibrosis in both groups (Sirius
red). E, F, SHR⫹Con-iRNA (F) presents a large area with positive ␣-smooth muscle actin (arrows) immunostaining, which indicates the presence of profibrotic cells (myofibroblasts) in cardiac interstitium, relative to the SHR⫹TRH-iRNA group (E). G, H,
Collagen III immunostaining in both groups. Note a large area of
positive immunostaining (arrows) in the SHR⫹Con-iRNA group
compared to SHR⫹TRH-iRNA. I, Bars represent the quantification of ECM expansion, ␣-smooth muscle actin, and collagen III
immunostaining areas in both groups.
treatment that prevents the increase in the myocyte diameter
and the ECM protein expression. These results are consistent
with the prevention of hypertrophy ratio increases in the
TRH-iRNA–treated group.
This work was supported by grant PICT 03-13862 from the
Agencia Nacional de Promocion Cientifica y Tecnológica
(ANPCyT) and grant PIP 11220090100636 from the National
Council of Research and Technology (CONICET).
Disclosures
None.
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Downloaded from http://hyper.ahajournals.org/ by guest on May 4, 2017
Cardiac Thyrotropin-Releasing Hormone Mediates Left Ventricular Hypertrophy in
Spontaneously Hypertensive Rats
Mariano L. Schuman, Maria S. Landa, Jorge E. Toblli, Ludmila S. Peres Diaz, Azucena L.
Alvarez, Samuel Finkielman, Leonardo Paz, Gabriel Cao, Carlos J. Pirola and Silvia I. García
Downloaded from http://hyper.ahajournals.org/ by guest on May 4, 2017
Hypertension. 2011;57:103-109; originally published online December 6, 2010;
doi: 10.1161/HYPERTENSIONAHA.110.161265
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1
ONLINE SUPPLEMENT
Cardiac Thyrotropin Releasing Hormone (TRH) Mediates Left Ventricular Hypertrophy in
Spontaneously Hypertensive Rats (SHR).
Mariano L Schuman, PhD1 ; Maria S Landa, PhD1,2; Jorge E Toblli, PhD,MD4; Ludmila S
Peres Diaz1; Azucena L Alvarez2; Samuel Finkielman, MD1; Leonardo Paz, MD3 ; Gabriel
Cao, MD4; CJ Pirola, PhD2 and Silvia I García, PhD1.
Departamento de Cardiología Molecular1, Departamento de Genética y Biología Molecular
de Enfermedades Complejas2 and Servicio de Anatomía Patológica3 Instituto de
Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires, Buenos Aires,
Argentina; IDIM-CONICET; Laboratory of Experimental Medicine, Hospital Alemán,
Buenos Aires, Argentina4.
“TRH and Cardiac Hypertrophy in SHR”
Correspondence Author:
Silvia I. García, PhD.
Departamento de Cardiología Molecular,
Instituto de Investigaciones Médicas Alfredo. Lanari, UBA
IDIM-CONICET
2
Materials and Methods
In vivo iRNA treatment:
Previously, we performed a dose-curve (20, 40, and 100 µg iRNA per injection), and as we
found the maximal downregulation effect at 40 ug we decided to use it as unique dose.
Forty µg iRNA/100 µL saline were injected in the left ventricle (LV) wall. All injections
were done under echography, the chests of the rats were shaved and cleaned. Gel was
liberally applied to the exterior body surface before the placement of the probe. The probe
was aligned with the needle and special care was taken to visualize the region of the
myocardial wall. Injection was divided in three different sites (from base to apex) to
surround the entire LV. iRNA oligonucleótidos were dissolved in saline. Systolic blood
pressure (SBP) by the plethismography-tail method and body weight were measured
weekly. All experiments were also performed blindly with respect to the treatment.
Specific stealth iRNAs from Invitrogen (CA, USA) were used, which present a chemical
modification resulting in lower toxicity and higher stability. The Block-iT TM iRNA
Designer software for the iRNA selection was used, and a mix of two double-strand
sequences against preproTRH (TRH-iRNA 498 and TRH-iRNA 138) and a mix of two
control iRNA sequences carrying mismatches compared with the specific ones (Con-iRNA
498 and Con-iRNA 138) were used as follows:
TRH-iRNA 498: S:5´ACGGUCCUGGUUACCAGAUUUCUUU3´ and
AS :5´AAAGAAA UCUGGUAACCAGGACCGU 3´;
TRH-iRNA 138: S:5` GAUCUUCACCCUAACUGGUAUCCCU 3´ and
AS: 5`AGGGAUACCAGUUAGGGUGAAGAUC 3`
Con-iRNA 498: S:5´ACGCCGUUGAUCCGAUAUUCUGUUU 3´ and
AS :5´AAACAGAAUAUCGGAUCAACGGCGU 3´;
Con-iRNA 138: S:5´GAUCACUAUCCGUCAUAUGCUCCCU 3´ and
AS :5´AGGGAGCAUAUGACGGAUAGUGAUC 3´
The screening of known rat genes from genomic databases of the National Center for
Biological Information using the Blast program indicates specificity of the sequence used in
the iRNA design and confirms their 100% homology with rat preproTRH gene.
The observed effects are not due to a nonspecific toxicity because the survival of animals
during the treatment was 100% and no sign of toxicity after decapitation and morphology
analysis was observed.
TRH content:
For peptide extraction, cardiac tissue samples were collected in acetic acid 2 mol/L, HCl
0.1 mol/L. All samples were boiled for 20 min and centrifuged at 10000 rpm, and the
supernatant was lyophilized. The residue was dissolved in the appropriate buffer for
measurement of TRH-like immunoreactivity by RIA or the mobile phase for the
identification of authentic TRH by high performance liquid chromatography. This
procedure and the RIA for TRH have been reported in detail 1. Briefly, the cross-reactivity
of the antiserum was 100% for p-Glu-His-Pro-NH2 (TRH), 15% for 3-methyl-TRH, 2.5%
3
for Glu-TRH, 0.03% for p-Glu-Glu-Pro-NH2, 0.02% for TRH-OH, and <0.002% for Glu,
His, Pro and cyclo(Hys-Pro). Standards or samples were incubated with 125I-TRH and antiTRH (1/8000) at 4°C overnight. The free hormone was pelleted using Carbon Dextran T70. All samples were assayed in duplicate. The minimum detectable amount was 5 pg.
Intra- and inter-assay coefficients of variation were less than 7.0 and 10.0 %, respectively,
along the standard curve concentrations used.
Quantitative real-time PCR:
Quantification of preproTRH, BNP, type III collagen, and TRH-RI mRNA expression was
performed using a real-time RT-PCR technique normalized by the ß-actin housekeeping
gene expression. Briefly, for cDNA synthesis, 2 µg of total RNA and 1 µg of random
hexamer primers (Promega Corp, WI, USA) were heated at 70°C for 5 min, followed by
incubation at 4ºC. After adding 5 uL of M-MLV RT 5X buffer (250 mmol/LTris-HCl, pH
8.3, 375 mmol/L KCl, 15 mmol/L MgCl2, 50 mmol/L DTT), 25 U RNasin (Promega Corp,
WI, USA), 1.25 uL of 10 mmol/L dNTPs (Invitrogen, CA, USA), and 200 U of MMLVreverse transcriptase (Promega Corp, WI, USA), the reaction was carried out at 37ºC for 1
h, followed by inactivation of the enzyme at 95ºC for 10 min.
Real-time PCR was performed using a BioRad iQ iCycler Detection System (BioRad
Laboratories, Ltd, CA, USA) with SYBER green fluorophore. Reactions (duplicates) were
carried out in a total volume of 20 uL. A 3-step protocol (95°C for 30 sec, 60°C, 64°C or
66°C annealing for 30 sec, 72°C for 40 sec) was repeated for 40 cycles. Rat (r) primers
(Invitrogen, CA, USA) were designed as follows:
r-preproTRH:F:5´GGTGCTGCCTTAGACTCCTG3´;
R:5´CTCCTCATCTGCCCATGAAT3´;
r-BNP: F:5´ATCCACGATGCAGAAGCTG3´; R:5´AGGCGCTGTCTTGAGACCTA3´;
r-CollgIII: F:5´GTCCAGGGATACGGGGTATG3´; R:
5´CAGATGGGCCAGGAGGAC3´;
r-ß-actin: F:5´TTCCTGGGTATGGAATCCTG3´; R:5´CAGCAATGCCTGGGTACAT3´ ;
r-Trh-RI: F:5’AAGCAGGTCACCAAGATGCT3’;
R:5’GTAAATCACCGGGTTGATGG3’
A melt curve analysis was performed following every run to ensure a single amplified
product for every reaction. The authenticity of the amplicons generated was confirmed by
their size in 2% agarose gel. In accordance with the literature, we did not find any
difference in the ß-actin used as housekeeping gene, between the experimental groups.
Pathology examination:
Hearts were perfused with saline solution through the abdominal aorta until they were free
of blood. Tissue was fixed in phosphate-buffered 10% formaldehyde (pH 7.2) and
embedded in paraffin. Three-micron sections were cut and stained with hematoxilin-eosin
(H&E), Masson’s Trichrome, and Sirius red. All observations in light microscopy were
performed using a Nikon E400 light microscope (Nikon Instrument Group, Melville, New
York, USA).
Immunohistochemical studies:
Immunolabeling of specimens was carried out using a modified avidin-biotin-peroxidase
technique (Vectastain ABC kit, Universal Elite, Vector Laboratories, California, USA).
Following deparaffinization and rehydration, the sections were washed in phosphate buffer
4
saline (PBS) for 5 min. Quenching of endogenous peroxidase activity was achieved by
incubating the sections for 30 min in 1% hydrogen peroxide in methanol. After washing in
PBS (pH 7.2) for 20 min, the sections were incubated with blocking serum for a further 20
min. Thereafter, the sections were rinsed in PBS and incubated with biotynilated universal
antibody for 30 min. After washing in PBS a final time, the sections were incubated for 40
min with Vectastain Elite ABC reagent (Vector Laboratories, California, USA) and
exposed for 5 min to 0.1% diaminobenzydine (Polyscience, Warrington, Pennsylvania,
USA) and 0.2% hydrogen peroxide in 50 mmol/L Tris buffer (pH 8). For the purpose of
evaluating the process of fibrosis, an anti-mouse α-smooth muscle actin monoclonal
antibody (sc-130617 Santa Cruz Biotechnology, Inc. CA) and a monoclonal antibody anticollagen type III (Biogenex, San Roman, CA), both at a dilution of 1:100, were used.
Morphological and quantitation analysis
All tissue samples were evaluated independently by two investigators without prior
knowledge of the group to which the rats belonged. All measurements were carried out
using an image analyzer, Image-Pro Plus version 4.5 for Windows (Media Cybernetics, LP,
Silver Spring, Maryland, USA). Histomorphometric evaluation of heart was carried out
according to the following schedule: extracellular matrix (ECM) expansion, numerical
density of myocytes and coronary capillaries (vessels < 8µ) within the confines of each of
the 20 random viewed at 400x magnification. In every heart, positive immunostaining for
α-smooth-muscle actin and collagen III was assessed and the data were expressed as
percentage per area.
Biochemical and pathology studies:
At the end of the treatment, the rats were weighted and the snout-tail long index was
measured before the animals were sacrificed by decapitation. Hearts were rapidly removed
and weighted. In some animals, cardiac tissues were separated (atria, left ventricle, right
ventricle, and septum) for TRH determination by RIA and for quantification of preproTRH,
brain natriuretic peptide (BNP), type III collagen, and TRH-R1 mRNA expression by realtime RT-PCR; others were used for pathology studies. LVH indexes were expressed as
heart to total body weight (HW/BW) and heart to snout-tail long-ratios.
Statistics:
Values were expressed as mean + SEM. All statistical analyses were performed using
absolute values and processed through Statistics, version 6.0 (Software, Inc, California,
USA). The assumption test to determine the Gaussian distribution was performed by the
Kolmogorov–Smirnov method. For parameters with Gaussian distribution, comparisons
between groups were carried out using one-way analysis of variance (ANOVA) followed
by Bonferroni’s test, Kruskal–Wallis test (nonparametric ANOVA), and Dunn’s multiple
comparison test when appropriate. In the case of two groups, comparison was performed
using t-test or Mann-Whitney test when appropriate. A p value of less than 0.05 was
considered significant.
References:
1- García SI, Dabsys SM, Martinez VN, Delorenzi A, Santajuliana D, Nahmod VE,
Finkielman S, Pirola CJ. Thyrotropin-releasing hormone hyperactivity in the preoptic area
of spontaneously hypertensive rats. Hypertension. 1995;26:1105-1110.
5
Table S1
Morphometric Variables
SHR+Con-iRNA
SHR+TRH-iRNA
p value
LVW cardiomyocyte diameter (µm)
37.7 ± 0.9
31.2 ± 0.7
< 0.01
9.8 ± 1.0
< 0.01
LVW cardiomyocyte density (number 5.6 ± 0.4
of cardiomyocyte /area)
LVW number of capillaries /area
11.7 ± 0.6
21.1 ± 1.1
< 0.01
LVW myocyte / capillary ratio
0.48 ± 0.03
0.46 ± 0.03
NS
IVS cardiomyocyte diameter (µm)
36.3 ± 1.2
29.9± 0.8
< 0.01
11.0 ± 1.0
< 0.01
IVS cardiomyocyte density (number of 6.1 ± 0.6
cardiomyocyte /area)
IVS number of capillaries /area
12.2 ± 0.7
20.6 ± 0.8
< 0.01
IVS myocyte / capillary ratio
0.51 ± 0.06
0.54 ± 0.06
NS
LVW: left ventricular wall
IVS : interventricular septum