1 1
Original Article
2 3 4 5
Sacubitril/valsartan inhibits cardiomyocyte hypertrophy in angiotensin II-induced hypertensive
6
mice independent of a blood pressure-lowering effect
7 8
Kohei Tashiro,
1Takashi Kuwano,
1Akihito Ideishi,
1Hidetaka Morita,
1Yoshiaki Idemoto,
19
Masaki Goto,
1,2Yasunori Suematsu,
1Shin-ichiro Miura
1,210 11
1
Department of Cardiology, Fukuoka University School of Medicine, 814-0180, Japan.
12
2
Department of Cardiology, Fukuoka University Nishijin Hospital, 814-8522, Japan.
13 14
Short Title: Anti-hypertrophic effects of ARNI in mice
15
Word count: 2603
16 17
Corresponding Author: Takashi Kuwano, Department of Cardiology, Fukuoka University School
18
of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan. Tel: 81-92-801-1011; Fax:
19
81-92-865-2692;
20
E-mail: [email protected]
21 22
2
Abstract
1
Background: Hypertensive left ventricular hypertrophy is associated with the risk of heart
2
failure, coronary heart disease and cerebrovascular disease. Although Sacubitril/Valsartan
3
(SAC/VAL), a first-in-class angiotensin receptor neprilysin inhibitor, reduces the risks of death
4
and hospitalization for patients with heart failure, its mechanism of action is not fully understood.
5
We hypothesized that SAC/VAL is superior to other conventional drugs in reducing cardiac
6
hypertrophy.
7
Methods: Male C57BL/6J mice were implanted with an osmotic pump containing
8
angiotensin II (Ang II). After 7 days of Ang II infusion, mice were also treated with either
9
SAC/VAL, Valsartan, Enalapril or vehicle alone each day for two weeks. Blood pressure
10
measurement was done weekly, and echocardiography was performed before and 3 weeks after
11
infusion of Ang II. Histological analyses were done using extracted heart to investigate cardiac
12
hypertrophy and fibrosis.
13
Results: Ang II markedly elevated blood pressures in all of the treatment groups, and
14
there were no differences in the degree of blood pressure reduction among the SAC/VAL-,
15
Valsartan- and Enalapril-treated groups. Echocardiography showed that SAC/VAL significantly
16
suppressed the increase in LV wall thickness and tended to decrease LV mass. In a histological
17
analysis, SAC/VAL inhibited Ang II-induced cardiomyocyte hypertrophy, and individual
18
cardiomyocytes in the SAC/VAL group were smaller than those in the Valsartan and Enalapril
19
groups. Although previous studies using animal models of heart failure have indicated that
20
SAC/VAL attenuates cardiac fibrosis, we found no supporting evidence in this setting.
21
Conclusion: SAC/VAL, Valsartan and Enalapril all attenuated cardiomyocytes hypertrophy
22
in a mouse model of Ang II-induced cardiac hypertrophy. Of note, SAC/VAL most strongly
23
suppressed hypertrophy in spite of similar blood pressure-lowering effects as Valsartan and
24
Enalapril. The present study suggests that SAC/VAL may have a beneficial effect on the early
25
stage of hypertensive heart disease.
26
3 1
Key Words: ARNI, Hypertension, Hypertrophy, Myocytes, Sacubitril
2 3
4
Introduction
1 2
Despite the availability of effective antihypertensive drugs, hypertension is still a major
3
risk factor for heart failure, myocardial infarction, stroke and kidney disease[1]. Indeed, more
4
than 40% of patients with hypertension are not controlled even when they receive two or more
5
drugs[2]. Continuous arterial hypertension results in increased cardiac afterload, and leads to left
6
ventricular (LV) wall thickening. LV hypertrophy has been associated with increased
7
cardiovascular (CV) risk even though it initially occurs as a compensatory mechanism to
8
minimize wall stress[3]. The renin-angiotensin system (RAS) plays an important role not only in
9
the systemic regulation of arterial pressure and blood volume, but also in regulation of the
10
growth of cardiomyocytes as an autocrine/paracrine factor[4], leading to LV hypertrophy. In fact,
11
chronic infusion of a subpressor dose of angiotensin II (Ang II) into mice induced cardiac
12
hypertrophy and fibrosis[5], and angiotensin-converting enzyme inhibitors (ACEi) and Ang II
13
receptor blockers (ARB) can reverse LV hypertrophy independent of their effect on blood
14
pressure[6-8]. Natriuretic peptides, including atrial natriuretic peptide (ANP) and b-type
15
natriuretic peptide (BNP), also counteract RAS activation through their diuretic, vasodilatory and
16
anti-mitogenic properties[9]. Inhibition of neprilysin augments the protective effect against RAS
17
through an increase in natriuretic peptides; thus, simultaneous neprilysin inhibition and
18
angiotensin receptor blockade can theoretically provide beneficial effects against RAS activation.
19
Sacubitril/Valsartan (SAC/VAL), a first-in-class angiotensin receptor neprilysin inhibitor (ARNI),
20
has been shown to reduce the risks of death and hospitalization for patients with heart failure[10].
21
Although patients receiving SAC/VAL had significantly lower blood pressure than those in the
22
ACEi Enalapril group in the PARADIGM-HF study, it did not affect the differences in event
23
rates. It has also been shown that SAC/VAL leads to sustained reduction of myocardial wall
24
stress and injury in surviving patients with heart failure[11]. These clinical findings suggest that
25
SAC/VAL may reduce LV hypertrophy independent of any lowering of blood pressure. Therefore,
26
5
we investigated the effect of SAC/VAL on LV remodeling in a mouse model of Ang II-induced
1
cardiac hypertrophy.
2 3
Materials and Methods
4
Animal Care
5
Animal experiments were performed according to institutional and governmental guidelines. The
6
protocol was approved by the Animal Care and Use Committee of Fukuoka University. All
7
procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of the
8
Institute of Laboratory Animal Resources.
9 10
Experimental protocol in vivo
11
Male C57BL/6J mice were purchased from Charles River Laboratories Japan, Inc. (Kanagawa,
12
Japan). Eight-week-old mice were weighed and anesthetized by the inhalation of 1.5%
13
isoflurane. An incision was made in the midscapular region, and mini-osmotic pumps (Alzet, CA,
14
USA) were implanted into each animal. The experimental groups received Ang II-loaded pumps
15
that delivered at a rate of 3.2 mg/kg/day continuously for 3 weeks. Ang II infusion at a rate of 0.5
16
mg/kg/day is sufficient to increase blood pressure in C57BL6 mice[12], while Ang II is widely
17
used at higher doses (1.4–3.2 mg/kg/day) to examine cardiac hypertrophy[13, 14].
18
Sham-operated animals underwent identical procedures, except that an osmotic pump with saline
19
was implanted. On the seventh day of Ang II infusion, mice were treated with either 60 mg/kg
20
SAC/VAL (salt complex at a 1:1 molar ratio), 30 mg/kg Valsartan, or 12 mg/kg Enalapril
21
dissolved in corn oil, or only corn oil every day for 2 weeks by oral gavage. The dosage of
22
SAC/VAL was determined from previous studies in rodents, all of which used around 60
23
mg/kg/day of SAC/VAL[15-17]. In addition, it has been reported that drug toxicity was observed
24
at 114 mg/kg/day SAC/VAL[18].
25 26
6
Measurement of blood pressure
1
Blood pressures were measured by a tail cuff-based MK-2000 (Muromachi Kikai Co., Ltd.,
2
Tokyo, Japan) before surgery and before death. Weekly blood pressures were also obtained
3
throughout the experiment to examine the time course of blood pressure elevation in response to
4
Ang II.
5 6
Echocardiographic analysis
7
All mice underwent blinded echocardiography before implantation of the osmotic mini-pump
8
and at the end of the experiment. Echocardiographic analysis was performed using a NEMIO
9
SSA-550A (Toshiba, Tokyo, Japan). Interventricular septum thickness diameter (IVSTd), left
10
ventricular posterior wall thickness diameter (LVPWd), left ventricular internal dimension in
11
diastole (LVDd) and heart rate (HR) were measured in M-mode at the level of the papillary
12
muscle. Echocardiographic LV mass was calculated (mg) as
13
[(LVDed+IVSWTh+PWTh)
3−LVDd3]×1.055, where 1.055 (mg/mm
3) is the density of the
14
myocardium[19]. LV mass was corrected for body weight and expressed as LV mass index
15
(LVMI).
16 17
Histological analysis
18
At the end of the experiment, all mice were euthanized under anesthesia. The heart tissue was
19
perfused with PBS, and then fixed with 4% paraformaldehyde and paraffin embedding. Sliced
20
samples were stained with Hematoxyline-Eosin and Picro-Sirius Red. For measurement, four
21
random high-power fields from each section were chosen and quantified in a blinded manner
22
using Image J software (National Institutes of Health, MD, USA). The cross-sectional area of
23
individual cardiomyocytes was analyzed quantitatively by morphometry of Hematoxylin-Eosin
24
stained sections. Four sections for each heart (the mean number of cardiomyocytes was 42 cells
25
per section) were counted. The extent of fibrosis was expressed as the ratio of Picro-Sirius Red
26
7
stained area to LV wall area.
1 2
Quantitative polymerase chain reaction (qPCR) analysis
3
Left ventricular apical regions were frozen in liquid nitrogen and stored at -80 ˚C. Total RNA
4
was extracted using a RiboPure RNA Purification Kit (Thermo Fisher Scientific Inc., MA, USA).
5
cDNA was produced using a RiverTra Ace qPCR RT Master Mix (TOYOBO Co., Ltd., Osaka,
6
Japan). qPCR was performed with a 7500 Fast Real‐Time PCR System (Applied Biosystems,
7
MA, USA) using a THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan). ANP, BNP and
8
transforming growth factor-β (TGF-β) were investigated. The primers used were forward primer
9
GGGGGTAGGATTGACAGGAT and reverse primer ACACACCACAAGGGCTTAGG for
10
mouse ANP, forward primer TCCTAGCCAGTCTCCAGAGC and reverse primer
11
CCTTGGTCCTTCAAGAGCTG for mouse BNP, and forward primer
12
GCTTCTAGTGCTGACGCCG and reverse primer GACTGGCGAGCCTTAGTTTG for mouse
13
TGF-β.
14 15
Statistical analysis
16
The statistical analysis was performed using SAS software, version 9.4 (SAS Institute, Cary, NC,
17
USA). The values are expressed as the mean and standard deviation. Group differences were
18
analyzed by using the unpaired T-test and the ANOVA test. Statistical significance was defined
19
as p-value <0.05.
20 21
Results
22
Changes in blood pressure by Ang II infusion
23
Ang II infusion (3.2mg/kg/day) gradually increased both systolic and diastolic blood pressures
24
compared with those in the saline-infused sham group, and these increases were significant at 3
25
weeks (Figure 1). Mice treated with SAC/VAL (60mg/kg/day), Valsartan (30mg/kg/day) or
26
8
Enalapril (12mg/kg/day) had similar levels of blood pressure throughout the experiment. Ang II
1
induced increases in blood pressures in all of the three treatment groups, and the blood pressure
2
values tended to be lower than those in the vehicle-treated control group, although these
3
differences were not significant.
4 5
Impact of SAC/VAL treatment on echocardiographic LV hypertrophy in Ang II-induced
6
hypertensive mice
7
We performed an echocardiographic analysis before Ang II infusion and immediately before we
8
euthanized the animals (Figure 2). Three weeks of Ang II infusion significantly increased IVSTd
9
and LVPWd, but did not alter LVDd. This suggests that Ang II led to concentric “pathological”
10
hypertrophy[20]. Treatment with SAC/VAL, but not Valsartan or Enalapril, suppressed the Ang
11
II-induced increase in LV wall thickness. There were also significant differences in IVSTd and
12
LVPWd between the vehicle control group and the SAC/VAL group at the end of the experiment.
13
Ang II infusion increased LV mass, and only treatment with SAC/VAL tended to inhibit this
14
increase (post LVMI; control group 5.6 ± 1.0 mg/g vs. SAC/VAL group 4.7 ± 0.6 mg/g, p =
15
0.07).
16 17
Impact of SAC/VAL treatment on cardiomyocyte size and cardiac fibrosis evaluated by a
18
histological analysis
19
To investigate the effect of SAC/VAL administration on Ang II-induced echocardiographic
20
hypertrophy, we assessed cellular growth, proliferation and fibrosis in the myocardium with
21
Hematoxylin-Eosin staining and Picro-Sirius Red staining. The mean cross-sectional area of
22
individual cardiomyocytes was enlarged after 3 weeks of Ang II infusion in the vehicle-treated
23
control group (Figure 3A). The increases in cardiomyocyte size induced by Ang II were
24
significantly attenuated by all three treatments, and the inhibitory effect in the SAC/VAL group
25
was significantly greater than those in the Valsartan and Enalapril groups (Figure 3B, the mean
26
9
area of individual myocytes was 249.5 ± 18.4, 304.8 ± 34.3 and 340.6 ± 41.2 µm
2, respectively).
1
In the vast majority of disease processes, cardiac hypertrophy coincides with fibrosis[21].
2
Therefore, we also examined the extent of cardiac fibrosis by Picro-Sirius Red staining (Figure
3
4A). Interstitial and perivascular fibrosis at the mid-transverse section area of the LV wall was
4
modestly increased by Ang II infusion compared to that in sham-treated mice. Remarkably, we
5
found no differences in the extent of fibrosis among the vehicle-control, SAC/VAL, Valsartan
6
and Enalapril groups (Figure 4B).
7 8
Effects of SAC/VAL treatment on hypertrophic and profibrotic gene expression induced by
9
Ang II
10
Transcriptional activation of several genes, including ANP and BNP, is one of the hypertrophic
11
responses to pressure overload[22, 23]. Therefore, we analyzed the mRNA levels of ANP and
12
BNP in the hearts after 3 weeks of Ang II infusion (Figure 5). Ang II significantly upregulated
13
the expression levels of the ANP gene compared with those in the sham-treated group. The
14
mRNA levels of BNP were also significantly upregulated by Ang II in the vehicle-control,
15
Valsartan and Enalapril groups, but not in the SAC/VAL group. TGF-β is known as a master
16
regulator of pro-fibrotic signaling, and the mRNA levels of TGF-β were upregulated by Ang II in
17
all of the groups. This is consistent with the finding at the cellular level (Figure 4) that the
18
expression levels of the pro-fibrotic TGF-β gene were similar among the vehicle-control,
19
SAC/VAL, Valsartan and Enalapril groups.
20 21
Discussion
22
The present study demonstrated that SAC/VAL, a first-in-class ARNI, prevented cardiac
23
hypertrophy in a mouse model of Ang II-induced cardiac hypertrophy. Regardless of similar
24
blood pressure levels, treatment with SAC/VAL significantly inhibited ventricular wall
25
thickening and cardiomyocyte enlargement compared with conventional therapy (ACEi enalapril
26
10
and ARB valsartan). Furthermore, the anti-hypertrophic effect of SAC/VAL was uncoupled from
1
cardiac fibrosis.
2
Echocardiographic LV hypertrophy is an independent predictor of CV events,
3
including CV death, coronary events, stroke and heart failure in patients with
4
hypertension[24-26]. Endomyocardial biopsy analysis also showed that cardiomyocyte
5
hypertrophy was an independent predictor of mortality for patients with heart failure, and more
6
than 50% of patients with a cardiomyocyte diameter over 17.7 µm died within 3 years[27].
7
Meanwhile, meta-analyses reported that LV mass reduction with pharmacological control of
8
blood pressure was associated with a 46% reduction in CV events for HT patients[28], and ACEi
9
were the most powerful antihypertensive drugs for reducing left ventricular mass, compared to
10
beta blockers, calcium channel blockers and diuretics[29]. In this study, attenuation of cardiac
11
hypertrophy by SAC/VAL was confirmed echocardiographically and histologically, over ACEi or
12
ARB treatment. Therefore, our results support the notion that SAC/VAL has the potential to be
13
the treatment of choice in terms of CV protection for patients with hypertension.
14
Although the detailed molecular mechanism by which this drug combination confers CV
15
protection is not fully characterized, the present study provides several interesting findings. First,
16
SAC/VAL significantly inhibited LV hypertrophy independent of blood pressure, suggesting a
17
direct rather than indirect effect on cardiac tissue. TGF-β has been proposed to act in an
18
autocrine/paracrine fashion between cardiomyocytes and fibroblasts to stimulate cardiac
19
remodeling[30], and the inhibition of Ang II/TGF-β signaling is a pivotal direct mechanism of
20
the antihypertrophic effect of ARBs[31]. In addition, natriuretic peptides also have some direct
21
effects on counteracting RAS signaling[9]. Thus, SAC/VAL may provide an augmented
22
inhibitory effect on ANG II/TGF-β signaling. On the other hand, the concomitant use of ARB
23
with either ACEi or direct renin inhibitor did not further decrease LV hypertrophy, which
24
suggests that simultaneous blocking of RAS signaling for cardiac hypertrophy might reach a
25
plateau[32, 33]. Interestingly, in this study, TGF-β mRNA levels were similar among the three
26
11
treatment groups, although significant differences were found in cardiac remodeling. This
1
suggests that SAC/VAL might affect LV remodeling not only by blocking Ang II/ TGF-β
2
signaling, but also via different pathways. A recent in silico analysis to clarify the details of the
3
mechanism of action of SAC/VAL may support our findings; specifically, sacubitril might
4
attenuate hypertrophy by inhibiting phosphatase and tensin homolog deleted from chromosome
5
ten (PTEN)[34]. In fact, loss of PTEN prevents the development of LV remodeling in response to
6
pressure overload, but not in response to Ang II[35]. Further studies are required to clarify the
7
association between SAC/VAL and PTEN.
8
Another intriguing finding of this report is that SAC/VAL administration prevented the
9
emergence of hypertrophy, which did not result from the attenuation of cardiac fibrosis. Unlike
10
previous reports in various rodent models of heart failure, which showed that ARNI ameliorates
11
LV remodeling by reducing cardiac fibrosis[15-18], we found significant differences in LV
12
hypertrophy along with a similar degree of cardiac fibrosis among the SAC/VAL, Valsartan and
13
Enalapril groups. Although 60 mg/kg/day SAC/VAL was not enough to decrease blood pressures
14
in this experiment, the dosing was based on previously published doses of SAC/VAL in rodents.
15
Therefore, this new finding can probably be attributed to the different animal model used. Less
16
interstitial and perivascular cardiac fibrosis (1-2% of the total left ventricle area) was observed in
17
our experiment, perhaps because of the relatively short-term exposure to Ang II. However,
18
validated hypertrophic markers, such as ANP and BNP[23], are significantly upregulated by Ang
19
II infusion, indicating that cardiomyocyte hypertrophy was pathological rather than physiological.
20
Therefore, our results suggest that SAC/VAL may have beneficial effects on the early stage of
21
cardiac remodeling, as well as in patients with chronic stable heart failure with reduced ejection
22
fraction[10]. Meanwhile, the upcoming TRANSITION trial will compare in-hospital initiation of
23
SAC/VAL to post-discharge initiation in patients with acute heart failure occurring as either de
24
novo heart failure or deterioration of chronic heart failure[36], and will hopefully shed some
25
light on the timing for when to initiate SAC/VAL.
26
12
In conclusion, SAC/VAL, Valsaltan and Enalapril all attenuated cardiomyocyte
1
hypertrophy in mice induced Ang II. Of note, SAC/VAL had the strongest inhibitory properties
2
independent of blood pressure-lowering effect and the progression of cardiac fibrosis. To the best
3
of our knowledge, our investigation is the first study to show that SAC/VAL has a beneficial
4
effect on the early stage of hypertensive heart disease.
5 6
Acknowledgement
7
None to declare
8 9
Financial Disclosure
10
None to declare
11 12
Conflict of Interest
13
None to declare
14 15
Authors Contribution
16
K.T., T.K., and S.M. conceived and planned the experiments. K.T.,T.K., A.I., H.M, Y.I.,M.G., and
17
Y.S. carried out the experiments. K.T., and T.K. took the lead in writing the manuscript. S.M.
18
supervised the project. All authors provided critical feedback and helped shape the research,
19
analysis and manuscript.
20 21
Data Availability
22
The authors declare that data supporting the findings of this study are available within the article.
23 24 25
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24 25
Figure legends
26
Figure 1.
27
Time course of systolic and diastolic blood pressure. Data are mean ± SD (n = 6-8 each). *p <
28
0.05 vs. sham-operated group.
29 30
Figure 2.
31
Echocardiographic analysis pre- Ang II infusion and post- 3 weeks of Ang II infusion.
32
16
Interventricular septum thickness diameter (IVSTd), left ventricular posterior wall thickness
1
diameter (LVPWd) and left ventricular mass index (LVMI). Data are mean ± SD (n = 6-8 each).
2
*p < 0.05 vs. sham-operated group,
†p < 0.05.
3 4
Figure 3.
5
Histological analyses for the cardiomyocytes of left ventricle. (A) Representative photographs of
6
higher magnification views of Hematoxyline-Eosin-stained heart sections. (B) Quantitative
7
analysis for the cross-sectional area of individual cardiomyocytes. Data are mean ± SD.
8
Sacubitril/Valsartan-treated group (SAC/VAL). *p < 0.05 vs. sham-operated group,
†p < 0.05 vs.
9
vehicle-treated control group.
10 11
Figure 4.
12
Histological analyses for the cardiac fibrosis. (A) Representative photographs of the Picro-Sirius
13
Red-stained whole heart sections. (B) Quantitative analyses for the left ventricular fibrosis.
14
Sacubitril/Valsartan-treated group (SAC/VAL). Data are mean ± SD.
15 16
Figure 5.
17
Relative expression of genes of interest by the quantitative polymerase chain reaction. Atrial
18
natriuretic peptide (ANP), B-type natriuretic peptide (BNP), transforming growth factor-β
19
(TGF-β) and Sacubitril/Valsartan-treated group (SAC/VAL). Data are mean ± SD (n = 6-8 each).
20
*p < 0.05 vs. sham-operated group.
21
Sham Control SAC/VAL
Valsartan Enalapril
A
B
Sham Control SAC/VAL
Valsartan Enalapril
A
B
n.s.
