Lactation is a Risk Factor of Postpartum Heart Failure in Mice with Cardiomyocyte-specific Apelin

20 Pages·2016·7.68 MB·English

JBC Papers in Press. Published on March 31, 2016 as Manuscript M115.699009
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.699009
Lactation is a risk factor of postpartum heart failure in mice with
cardiomyocyte-specific apelin receptor (APJ) overexpression.

Kazuya Murata‡, Junji Ishida‡, §, Tomohiro Ishimaru§, Hayase Mizukami§, Juri Hamada‡,
Chiaki Saito§, and Akiyoshi Fukamizu‡, §

‡Life Science Center, Tsukuba Advanced Research Alliance, §Graduate School of Life and
Environmental Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan

Running title: Postpartum heart failure induced by APJ and lactation

Address all correspondence and requests for reprints to: Akiyoshi Fukamizu, Ph.D.
Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Ten-noudai,
Tsukuba, Ibaraki 305-8577, Japan.
Phone/Fax: [phone]
E-mail: [email protected]

D
Keywords: G protein-coupled receptor (GPCR), heart, heart failure, cardiac hypertrophy, pregnancy, o
w
cardiomyopathy, APJ, lactation nlo
a
de
d
fro
m
Abstract mice. Furthermore, we found that lactating h
G protein-coupled receptor APJ and its APJ-TG mice showed impaired myocardial ttp
ligand apelin are highly expressed in angiogenesis and imbalance of pro- and ://w
w
w
cardiovascular tissues, and are associated with anti-angiogenic gene expression in the heart. .jb
the regulation of blood pressure and cardiac These results demonstrate that overexpression of c.o
function. Although accumulating evidence APJ in cardiomyocytes has adverse effects on brg/
suggests that APJ plays a crucial role in the cardiac function in male and non-pregnant mice, y g
u
heart, it remains unclear whether upregulation of and lactation contributes to the development of es
t o
APJ affects cardiac function. Here, we postpartum cardiomyopathy in the heart with n
A
generated cardiomyocyte-specific APJ APJ overexpression. p
overexpressing (APJ-TG) mice, and investigated ril 2
, 2
the cardiac phenotype in APJ-TG mice. Male Introduction 0
1
9
and non-pregnant APJ-TG mice showed cardiac Pregnancy and lactation are essential
hypertrophy, contractile dysfunction, and processes in the reproduction of mammals, and
elevation of B-type natriuretic peptide (BNP) induce marked changes in systemic hormone
gene expression in the heart, but not cardiac and hemodynamic status, consequently affecting
fibrosis and symptoms of heart failure, including cardiac function (1, 2). Pregnant women show
breathing abnormality and pleural effusion. We increases in circulating blood volume, heart rate,
further examined the influence of APJ and cardiac output to provide sufficient blood to
overexpression in response to physiological their fetuses (3). It has been reported that
stress induced by pregnancy and lactation in the elevation of cardiac output and adaptive
heart. Interestingly, in female APJ-TG mice, hypertrophy are observed in lactating rats for
increment of pregnancy-lactation cycles supplying blood to mammary gland (4-6).
exacerbated cardiac hypertrophy and systolic Furthermore, we previously reported that
dysfunction, and induced cardiac fibrosis, lung lactation causes contractile dysfunction in
congestion, pleural effusion, and abnormal pregnancy-associated hypertensive mice (7).
breathing, showing that APJ-TG mice develop Thus, pregnancy and lactation are closely
postpartum cardiomyopathy. We showed that related to alteration of cardiac function.
lactation, but not parturition, was critical for the Although maternal cardiac function is
onset of postpartum cardiomyopathy in APJ-TG normally maintained throughout peripartum
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
period, in some women, heart failure of increased APJ expression on the cardiac
unknown etiology occurs between the last function, we generated cardiomyocyte-specific
month of pregnancy and the early postpartum, APJ overexpressing (APJ-TG) mice.
known as peripartum cardiomyopathy, which is Surprisingly, we found that female APJ-TG
especially called postpartum cardiomyopathy in mice develop severe heart failure in the
the case of developing heart failure during postpartum period, and lactation is a key factor
postpartum period (8, 9). Several factors, such in the pathogenesis of heart failure in
as viral infection, autoimmune responses, and postpartum APJ-TG mice.
hypertensive complications in pregnancy, have
been considered as triggers of peripartum Experimental procedures
cardiomyopathy (10-12). Recent works have Animals
demonstrated the existence of familial form of Human APJ cDNA (30) was subcloned into
peripartum cardiomyopathy (13-15), while α-myosin heavy chain (αMHC)
formerly peripartum cardiomyopathy was promoter-containing expression vector (kindly
defined as non-familial form of cardiomyopathy. gifted from Professor Jeffrey Robbins,
Moreover, the studies using genetically Cincinnati Children's Hospital Medical Center,
engineered mouse models revealed that Ohio, USA) (31). The linearized DNA (10 kb)
cardiomyocyte-specific overexpression of Gαq, was microinjected as a transgene into pronuclei D
o
one of the G-protein, induces marked of eggs from C57BL/6J mice. Mice were w
n
lo
cardiomyocyte apoptosis in the peripartum genotyped by Southern blotting. Briefly, the a
d
e
period, resulting in peripartum cardiomyopathy genomic DNA was prepared from tail of mice d
(16-18). Other studies have shown that and 1 µg of DNA was digested with EcoRI and from
cardiomyocyte-specific deletion of STAT3 or BglII. Digested DNA was separated by 0.7% http
PGC1α (peroxisome proliferator-activated agarose gel electrophoresis, transferred to a ://w
w
receptor gamma, coactivator 1 alpha) in mice positively charged nylon membrane using w
causes impaired cardiac angiogenesis, and leads alkaline buffer, and hybridized with [α-32P] .jbc
.o
to onset of postpartum cardiomyopathy (19, 20). dCTP-labeled probe for 5’-side of mouse APJ rg
Thus, pathogenesis of peripartum coding sequence (APJ probe, see Fig. 1A). This by/
g
cardiomyopathy is highly complex, and is not probe can recognize both mouse and human APJ u
e
s
fully understood. gene because of the high homology of DNA t o
n
APJ (also known as apelin receptor, APLNR, sequence. After washing and drying, membrane A
p
or AGTRL1) is one of the G-protein coupled was exposed to imaging plate. Image was ril 2
receptor, and is highly expressed in obtained using Typhoon 8600 and ImageQuant , 2
0
1
cardiovascular tissues (21, 22). It has been software (GE Healthcare). To analyze cardiac 9
reported that APJ and its ligand apelin are function, echocardiography was performed as
involved in the regulation of blood pressure, previously described (7). Animal experiments
angiogenesis, and maintenance of cardiac were performed in a humane manner and
function (21-24). Apelin administration approved by the Institutional Animal
increases cardiac contractility in isolated Experiment Committee of the University of
working heart models (25). Furthermore, apelin Tsukuba. Experiments were conducted in
knockout (KO) mice and APJ-KO mice show accordance with the Regulation of Animal
reduced cardiac contractility (26, 27). In Experiments of the University of Tsukuba and
addition to positive inotropic effect, the the Fundamental Guidelines for Proper Conduct
apelin-APJ system also has cardioprotective of Animal Experiments and Related Activities
effects against ischemia-reperfusion injury and in Academic Research Institutions under the
anticancer drug-induced cardiotoxicity (28, 29). jurisdiction of the Ministry of Education,
Although several lines of evidence suggest Culture, Sports, Science and Technology of
critical roles of the apelin-APJ system in the Japan.
heart, the effect of increased APJ expression on
cardiac function is not elucidated. Histological analysis
In this study, to investigate the effect of Harvested hearts were fixed in 4%
2
paraformaldehyde for 48 hours at 4ºC, washed Biosciences) for 60 min at room temperature.
with PBS, dehydrated, and embedded in paraffin. After incubating biotinylated anti-Rat IgG
Hematoxylin-Eosin (HE) and Masson’s antibody (#BA-4001, Vector Laboratories,
trichrome stains were performed as previously Burlingame, CA) for 30 min at room
described (29, 7). Immunohistochemistry for temperature, secondary antibodies were detected
APJ was carried out as previously described using TSA biotin system (#NEL700A,
(29). Briefly, fresh-frozen hearts were sectioned PerkinElmer) according to the manufacturer’s
into 10 µm, and dried at room temperature. instructions. To visualize plasma membrane and
Heart sections were fixed in 4% nuclei, sections were stained with
paraformaldehyde for 10 min at room CF594-conjugated WGA and Hoechst 33258.
temperature, blocked in 5% BSA for 30 min at Images were obtained using BZ-9000
room temperature. After endogenous (BIOREVO, Keyence, Osaka, Japan). The
avidin-biotin blocking (#415041, Nichirei number of capillaries per fifty cardiomyocytes
Biosciences, Tokyo, Japan), sections were was determined in ten different randomly
incubated with anti-APJ antibody (1:10, rabbit chosen areas using ImageJ software.
polyclonal, homemade) at 4ºC overnight.
Sections were washed in 0.5 M NaCl/0.05% Terminal
Tween-20/PBS (-) solution three times, and deoxynucleotidyltransferase-mediated D
o
incubated with biotinylated donkey anti-rabbit dUTP-biotin nick end labeling (TUNEL) w
n
antibody (#111-065-144, Jackson assay loa
d
e
ImmunoResearch Laboratories, West Grove, TUNEL assay was performed using an in d
PA) for 30 min at room temperature. For situ Cell Death Detection Kit, TMR red from
detecting biotinylated-antibody, sections were (#12156792910, Roche Diagnostics, Mannheim, http
incubated with CF488A streptavidin conjugate Germany) according to the manufacturer’s ://w
(#29034, Biotium, Hayward, CA) for 30 min at instructions. Briefly, deparaffinized heart ww
room temperature. To visualize plasma sections (5 µm) were incubated with 20 µg/ml .jb
c
membrane and nuclei, we used CF594 wheat proteinase K for 15 min at 37ºC. DNA .org
germ agglutinin (WGA, #29023, Biotium) and fragments were labeled with b/
y
g
Hoechst 33258. Using confocal laser scanning tetramethylrhodamine-dUTP using TdT for 1 h u
e
s
microscope (FLUOVIEW FV10i, Olympus, at 37ºC. For nuclear counter staining, sections t o
n
Tokyo, Japan), we obtained fluorescence images. were stained with Hoechst 33258. Fluorescence A
p
For measuring cross sectional areas, images were acquired using BZ-9000. The ril 2
deparaffinized cardiac sections were stained numbers of TUNEL positive cells were , 2
0
with CF594-conjugated WGA. Fluorescence determined in ten random fields using a BZ-II 19
images were obtained using confocal analyzer (Keyence). Data were represented as
microscope and analyzed ImageJ software percentage of TUNEL positive cells per total
(http://imagej.nih.gov/ij/). Fifty cardiomyocytes number of nuclei.
per section were evaluated.
Northern blotting
Capillary density Total RNA was extracted from frozen heart
For measuring capillary density, tissues using ISOGEN (#311-02501, Nippon
deparaffinized cardiac sections were treated Gene, Tokyo, Japan). After glyoxylation of
with 20 µg/ml proteinase K for 30 min at 37ºC. RNA, 10 µg of total RNA were separated by
To inactivate endogenous peroxidase, sections 1.2% agarose gel electrophoresis, transferred to
were treated with 3% hydrogen peroxide in a positively charged nylon membrane, and
methanol for 15 min at room temperature. hybridized with [α-32P] dCTP-labeled APJ
Sections were blocked with tyramide signal probe. After washing and drying, membrane
amplification (TSA) blocking reagent (#FP1020, was exposed to imaging plate. Image was
PerkinElmer, Waltham, MA) for 30 min at room obtained using Typhoon 8600 and ImageQuant
temperature and incubated with anti-CD31 software (GE Healthcare).
antibody (1:50, Rat polyclonal, #550274, BD
3
Quantitative real-time (qRT) PCR analysis mPdgfb; Forward
qRT-PCR was performed as previously 5’-CTCCGTAGATGAAGATGGGCC-3’
described (7). Expression levels for ANP (Nppa), mPdgfb; Reverse
BNP (Nppb), collagen I (Col1a1), α-myosin 5’-AGCTTTCCAACTCGACTCCG-3’
heavy chain (Myh6), and β-myosin heavy chain mThbs1; Forward
(Myh7) were determined as the number of 5’-CTCGGGGCAGGAAGACTATG-3’
transcripts relative to those of Gapdh. Levels of mThbs1; Reverse
Vegfa, Angpt1, Angpt2, Pdgfa, Pdgfb, Thbs1, 5’-TGGGCTGGGTTGTAATGGA-3’
and Thbs2 were determined as the number of mThbs2; Forward
transcripts relative to those of Hprt gene. Primer 5’-CTGGCATCGCTGTAGGTTTC-3’
sequences are shown below. mThbs2; Reverse
mNppa; Forward 5’-CCTGCTTCCACATCACCAC-3’
5’-GGTAGGATTGACAGGATTGGAG-3’ mHprt; Forward
mNppa; Reverse 5’-CTGGTTAAGCAGTACAGCCCC-3’
5’-GCAGAATCGACTGCCTTTTC-3’ mHprt; Reverse
mNppb; Forward 5’-TCAAATCCAACAAAGTCTGGCCT-3’
5’-GGGCTGTAACGCACTGAAG-3’
mNppb; Reverse D
o
5’-ACTTCAAAGGTGGTCCCAGAG-3’ Western blotting wn
mCol1a1; Forward Mouse hearts were harvested and loa
d
e
5m’C-GoAl1Ta1G;G ATTCCCGTTCGAG-3’ Reverse ismtomraegdei aatte -ly8 0ºCfr.o Czeanr diainc tilsisquueisd wenrietr opgoewnd erfeodr d from
5’-GCTGTTCTTGCAGTGATAGGTG-3’ by a multi-beads shocker (Yasui Kikai, Osaka, http
mMyh6; Forward Japan), and homogenized in RIPA buffer ://w
5’-CAAGCTCACTTGAAGGACACC-3’ containing phosphatase and protease inhibitors ww
mMyh6; Reverse (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, .jb
c
5’-CACGATGGCGATGTTCTC-3’ 1% NP-40, 0.5% sodium deoxycholate, 0.1% .org
mMyh7; Forward SDS, 1 mM DTT, 1 mM sodium orthovanadate, by/
5’-AACCAGACGGCACTGAAGAG-3’ 10 mM sodium fluoride, 20 mM gu
e
s
mMyh7; Reverse β-glycerophosphate, 2.1 µg/ml aprotinin, 1 t o
n
5’-TGCCCACTTTGACTCTAGGATG-3’ µg/ml leupeptin, 1 mM phenylmethanesulfonyl A
p
mGapdh; Forward fluoride). After incubation for 30 min, samples ril 2
5’-TCACTGGCATGGCCTTCC-3’ were centrifuged at 14,000 rpm for 15 min at , 2
0
1
mGapdh; Reverse 4ºC. The supernatant was transferred to new 9
5’-CAGGCGGCACGTCAGATC-3’ tubes, and protein concentration was determined
mVegfa; Forward using Protein Assay Dye Reagent Concentrate
5’-ACCCTGGTGGACATCTTCCA-3’ and Protein Standard I (#500-0005 and
mVegfa; Reverse #500-0006, Bio-Rad, Hercules, CA). Samples
5’-TCATCGTTACAGCAGCCTGC-3’ were mixed with 2x Laemmli sample buffer
mAngpt1; Forward (100 mM Tris-HCl, 2% SDS, 100 mM DTT
5’-CCGAGCCTACTCACAGTACGA-3’ 20% glycerol, 0.01% bromophenol blue), and
mAngpt1; Reverse incubated for 5 min at 99ºC. Protein samples
5’-CTGCTGTCCCTGTGTGACCTT-3’ (50-100 µg) were loaded and subjected to
mAngpt2; Forward SDS-PAGE, and transferred onto PVDF
5’-ACCTCGCTGGTGAAGAGTCC-3’ membrane. Membranes were blocked with 5%
mAngpt2; Reverse nonfat dry milk in Tris-buffered saline/Tween
5’-CTGGTTGGCTGATGCTACTTATTTT-3’ 20 (TBS-T) for 1 hour at room temperature.
mPdgfa; Forward Primary antibodies were diluted in 1% nonfat
5’-GGAGGAGACAGATGTGAGGTG-3’ dry milk in TBS-T, and incubated with
mPdgfa; Reverse membranes at 4ºC overnight. Primary antibodies
5’-GGAGGAGAACAAAGACCGCA-3’ used in this study included: anti-STAT3 (1:1000,
4
#610189, BD Biosciences); We generated two lines of transgenic mice
anti-phospho-STAT3 (Tyr705) (1:500, #9131, overexpressing the human APJ gene in
Cell Signaling Technology, Danbers, MA); cardiomyocytes using the αMHC promoter (Fig.
anti-Cathepsin D (1:1000, ab75852, abcam, 1A). Southern blot analysis revealed transgene
Cambridge, UK); anti-PGC1α (1:500, sc-13067, integration into genomic DNA of line 25 and
Santa Cruz Biotechnology, Dallas, TX); line 37 transgenic mice (Fig. 1B).
anti-phospho-AKT (Ser473) (1:500, #4060, Cell Transgene-derived human APJ mRNA was
Signaling Technology); anti-AKT (1:1000, detected in the heart of both line 25 and line 37
#2920, Cell Signaling Technology); mice (Fig. 1C). Unexpectedly, line 37
anti-phospho-ERK1/2 (Thr202/Tyr204) (1:500, homozygous transgenic mice showed rectal
#9101, Cell Signaling Technology); anti-ERK2 prolapse with high frequency (data not shown).
(1:500, #05-157, Millipore); anti-GAPDH Therefore, we have shown data from line 25
(1:2000, #05-50118, American Research transgenic mice in subsequent experiments
Products, Waltham, MA); anti-α-Tubulin unless otherwise noted. Next, we examined APJ
(1:5000, #T5168, SIGMA). Membranes were protein expression in the heart by
washed in TBS-T, and incubated with immunohistochemistry. Consistent with our
horseradish peroxiase-linked secondary previous report (29), in WT mice, APJ protein
antibody (GE Healthcare) diluted in 0.5% nonfat was expressed in plasma membrane of D
o
dry milk in TBS-T for 1 hour at room cardiomyocytes in a patchy fashion (Fig. 1D, wn
lo
temperature. After secondary antibody arrowheads). In the heart of homozygous APJ a
d
e
ainncdu bvaitsiuoanl,i zmede musbirnagn eS uwpaesr Swigansahle dW eins t TFBemS-tTo tdreatnescgteedn ici n mthicee , whinotleen spel asmfluao rmesecmenbcrea new aosf d from
Chemiluminescent Substrate (#34094, Thermo cardiomyocytes, but not in the coronary artery http
Fisher Scientific, Waltham, MA). (Fig. 1D). These data demonstrate that APJ is ://w
Chemiluminescence signals were detected using overexpressed in cardiomyocytes and is ww
a Fuji LAS-3000 imaging system (Fujifilm, localized to plasma membrane in the heart of .jb
c
Tokyo, Japan). APJ-TG mice. .org
by/
g
Plasma apelin measurement Overexpression of APJ causes cardiac u
e
s
Plasma samples were collected from blood hypertrophy and contractile dysfunction in t o
n
of non-pregnant and postpartum female mice by male mice. A
p
centrifugation at 3,000 rpm, frozen immediately, APJ-TG mice were born at a normal ril 2
and stored at -80ºC. Plasma apelin levels were Mendelian ratio (data not shown), and body , 2
0
determined by using an apelin-12 EIA kit weight was comparable between male WT and 19
(#EK-057-23, Phoenix Pharmaceuticals, APJ-TG mice at ages three and six months (Fig.
Burlingame, CA) according to the 2A). On the other hand, heart weight (HW) and
manufacturer’s protocol. heart weight per body weight ratio (HW/BW)
were increased in male APJ-TG mice compared
Statistical analysis with WT mice at ages three and six months (Fig.
Statistical analysis was performed using 2, B and C). Echocardiographic analysis
GraphPad Prism 5 (GraphPad Prism Software, revealed that male APJ-TG mice exhibited
La Jolla, CA). The data were analyzed with reduction of fractional shortening and elevation
Student’s t-test, one-way ANOVA with Tukey’s of left ventricular internal dimension (LVID)
post-hoc test, two-way ANOVA followed by relative to WT mice (Fig. 2, D-F, and
Bonferroni multiple comparison test. Significant supplemental Video S1). Although there were
differences were defined as P < 0.05. no significant differences in diastolic left
ventricular anterior and posterior wall thickness
Results (LVAW and LVPW) between WT and APJ-TG
mice, slight reductions in systolic LVAW and
Establishment of cardiomyocyte-specific APJ LVPW were observed in APJ-TG mice
overexpressing mice. compared to WT mice at the age of six months
5
(Fig. 2, G-J). Macroscopic and histological Preg). At four weeks after parturition (four
analyses showed that cardiac hypertrophy and weeks-postpartum, referred to as 4W-PP in
enlargement of cardiac chamber were occurred figures), in WT mice, which had finished
in APJ-TG mice, while no obvious fibrosis was breastfeeding their pups, showed significant
seen in six-month-old WT and APJ-TG mice increases in HW and HW/BW compared with
(Fig. 2K). There were no significant changes in non-pregnant WT mice (Fig. 3, A-D, Parity 1),
mean cross sectional areas of myofibers while their fractional shortening was maintained
between WT and APJ-TG mice (Fig. 2L). These (Fig. 3, E, F, and supplemental Video S2, Parity
data indicate that APJ-TG mice develop 1). By contrast, APJ-TG mice exhibited
eccentric cardiac hypertrophy. Furthermore, as reduction of cardiac contractility with increases
compared with WT mice, cardiac BNP (Nppb) in HW and HW/BW at four weeks postpartum
and β-myosin heavy chain (Myh7) gene (Fig. 3, A-F, and supplemental Video S2, Parity
expression levels were significantly increased in 1). The cross sectional areas of myofibers were
APJ-TG mice at ages three and six months (Fig. increased in both WT and APJ-TG mice at four
2M). Collagen I (Col1a1) expression was weeks postpartum compared with non-pregnant
significantly increased in three-month-old mice, while there were no significant changes
APJ-TG mice, and tended to be increased in between WT and APJ-TG mice (Fig. 3G).
six-month-old APJ-TG mice compared with WT Next, we investigated the effect of D
o
mice (Fig. 2M). There are no significant subsequent pregnancy and lactation on cardiac w
n
differences in ANP (Nppa) and α-myosin heavy function. In WT mice, subsequent load
e
cWhaTi na n(dM AyhP6J)- TgGe nme iceex p(Freisgs. io2nM )l.e vTehlse seb ertewsueletns pwrheegrneaansc Hy-Wla/cBtaWtio ann d fcryacclteios nali nschroeratesendin g wHeWre, d from
suggest that APJ overexpression in not affected, indicating that WT mice showed http
cardiomyocytes causes pathological cardiac physiological hypertrophy. (Fig. 3, A-F, and ://w
hypertrophy and contractile dysfunction in male supplemental Video S2, Parity 2, 3). However, ww
mice. However, it seems likely that these in APJ-TG mice, further increase in HW/BW .jb
c
pathological cardiac phenotypes are not serious, and reduction of fractional shortening were .org
since APJ-TG mice did not show cardiac induced by repeated pregnancy-lactation cycles by/
fibrosis, elevation of ANP expression, and other (Fig. 3, A-F, and supplemental Video S2, Parity gu
e
s
features of heart failure, such as abnormal 2, 3). Since fractional shortening of six t o
n
breathing and pleural effusion. More month-old non-pregnant APJ-TG mice was A
p
importantly, cardiac hypertrophy and contractile similar to that of two month-old mice (Fig. 3F), ril 2
dysfunction did not get worse by aging in male decrease of cardiac contractility in parous , 2
0
APJ-TG mice (Fig. 2, C and D). APJ-TG mice was not due to aging stress. 19
Moreover, APJ-TG mice, which had
APJ overexpression induces postpartum experienced pregnancy and lactation more than
cardiomyopathy in female mice. two times, exhibited lung congestion and
Pregnancy and lactation are considered as cardiac fibrosis (Fig. 3, H and I). In addition,
physiological stress on the maternal heart (1, 2, abnormal breathing and pleural effusion were
19). Next, to investigate whether APJ observed in APJ-TG mice that experienced three
overexpression influences the response to times pregnancy-lactation cycles (supplemental
physiological stress during pregnancy and Video S3, and Fig. 3J). These results indicate
lactation, we analyzed female APJ-TG mice. that physiological stress induced by pregnancy
Non-pregnant female APJ-TG mice developed and lactation causes postpartum cardiomyopathy
cardiac hypertrophy and contractile dysfunction, in APJ overexpressing mice.
as well as male APJ-TG mice (Fig. 3, A-F, and
supplemental Video S2, NP). Although HW/BW Lactation induces postpartum
was decreased in pregnant mice because of body cardiomyopathy in APJ-TG mice.
weight gain, pregnancy did not affect HW and Although we found that APJ-TG mice show
fractional shortening in both WT and APJ-TG cardiomyopathy in the postpartum period, it
mice (Fig. 3, A-F, and supplemental Video S2, remained unclear whether parturition or
6
lactation or both induce heart failure in APJ-TG These data demonstrated that lactation is critical
mice. To determine the critical inducer of for the onset of postpartum cardiomyopathy in
postpartum cardiomyopathy in APJ-TG mice, APJ overexpressing mice. Next, we investigated
we analyzed postpartum dams with or without whether lactation induces apelin expression,
lactation. As mentioned above, lactation induced resulting in activation of overexpressed APJ.
cardiac hypertrophy in both WT and APJ-TG However, compared with non-pregnant mice
mice, while dams without lactation did not show and mice without lactation, plasma apelin levels
elevation of HW/BW compared with did not increase in both WT and APJ-TG mice
non-pregnant mice (Fig. 4, A and B). Fractional with lactation at two and four weeks postpartum
shortening of APJ-TG mice without lactation (Fig. 4F). Moreover, lactation did not increase
was significantly higher than that of APJ-TG cardiac apelin gene (Apln) expression in both
mice with lactation, and was comparable to that WT and APJ-TG mice (Fig. 4G). At four weeks
of non-pregnant APJ-TG mice (Fig. 4C). It postpartum, apelin expression was significantly
should be noted that, in lactating APJ-TG mice, decreased in APJ-TG mice with lactation
cardiac contractility at four weeks postpartum compared to WT mice and non-pregnant
did not show further decrease compared to the APJ-TG mice (Fig. 4G).
level at three weeks postpartum (Fig. 4C,
3W-PP, 4W-PP Lac (+)). Considering that pups APJ overexpression impairs cardiac D
o
began to eat food pellets rather than drinking angiogenesis in the postpartum period. w
n
milk around three weeks postpartum, cardiac Previous studies have demonstrated that the loa
d
e
flaucntcattiioonn ofrfo AmP tJh-rTeGe tdoa mfosu rw wase enkost paoffsetpctaerdtu mby. dcaerfdicioiemncyyo cyotfe s STcaAuTse3s oirm pPaGirCed1 α mgyeonceasr diianl d from
These lactation-dependent cardiac hypertrophy angiogenesis in the postpartum period, resulting http
and reduction of cardiac contractility were also in peripartum cardiomyopathy (19, 20). ://w
observed in line 37 APJ-TG mice (Fig. 4, B and Therefore, we next investigated the capillary ww
C). Furthermore, significant increases in cardiac density and protein expression levels of STAT3 .jb
c
ANP, BNP, and collagen I gene expression were and PGC1α in the heart of APJ-TG mice with .org
observed in APJ-TG mice with lactation, but not lactation. Although there was no difference in by/
in APJ-TG mice without lactation (Fig. 4D). In cardiac capillary number between non-pregnant gu
e
s
WT mice, only BNP gene expression was WT and APJ-TG mice, lactating APJ-TG mice t o
n
elevated by lactation (Fig. 4D). Although the had reduced capillary number compared with A
p
reason why BNP gene expression is increased in WT mice, indicating that cardiac angiogenesis is ril 2
WT mice by lactation is unknown, it is possible impaired in postpartum APJ-TG mice (Fig. 5A). , 2
0
that the cardioprotective effect of BNP through In the postpartum period, STAT3 protein levels 19
its receptor GC-A signaling (32) may play a role were similar between WT and APJ-TG mice at
in maintaining cardiac function in postpartum two and four weeks postpartum (Fig. 5B,
period in WT mice. middle). It has been reported that STAT3 is
It has been reported that elevation of phosphorylated in the heart of postpartum mice,
apoptosis in cardiac tissue is observed in mouse and its level is decreased after weaning of their
models of peripartum cardiomyopathy, such as pups (19). Phosphorylation of STAT3 is
Gαq transgenic mice and important for its nuclear localization and
cardiomyocyte-specific STAT3 deficient mice activation (33). At two weeks postpartum,
(16, 19). Therefore, we further investigated the phosphorylated STAT3 was detected in both
levels of apoptosis in postpartum APJ-TG mice. WT and APJ-TG mice at comparable levels (Fig.
As shown in Fig. 4E, the number of TUNEL 5B, top). Furthermore, cardiac STAT3
positive cells was significantly increased in deficiency causes excessive oxidative stress in
APJ-TG mice with lactation at four weeks the postpartum period, and leads to upregulation
postpartum compared with non-pregnant of cathepsin D protein levels (19). However,
APJ-TG mice, but not in APJ-TG mice without cathepsin D protein levels were not increased in
lactation, while lactation did not affect the APJ-TG mice as compared with WT mice at
number of TUNEL positive cells in WT mice. two weeks postpartum (Fig. 5C). There were no
7
differences in protein expression levels of cardiomyopathy in APJ overexpressing mice.
PGC1α between WT and APJ-TG mice at two It has been reported that apelin peptide has a
weeks postpartum (Fig. 5D). positive inotropic effect, and both apelin and
We next examined the alterations in other APJ are essential for maintenance of cardiac
signaling pathways. We previously reported that contractility in mice (25-27, 29). On the other
apelin treatment induces transient AKT and hand, we showed that APJ overexpression
ERK1/2 phosphorylation in HEK293T cells contributed to contractile dysfunction with
stably expressing human APJ gene (30). A eccentric hypertrophy in male and non-pregnant
recent work has revealed that constitutive AKT female mice. This discrepancy may be explained
activation in the heart also contributes to the by activation of sodium-calcium exchanger
development of postpartum cardiomyopathy (NCX). NCX has been shown to be involved in
with decreased capillary density (34). Thus, we the positive inotropic effect of apelin in working
investigated AKT and ERK1/2 phosphorylation rat heart model (25). Interestingly, cardiac
levels in the heart of APJ-TG mice. However, overexpression of NCX1, which is mainly
phosphorylation levels of AKT and ERK1/2 expressed in cardiac muscle, induces eccentric
were comparable in the heart of WT and hypertrophy and decrease in cardiac contractility
APJ-TG mice (Fig. 5D). These results suggest (40). Crucially, postpartum homozygous NCX1
that APJ-TG mice exhibit postpartum transgenic mice show significant reduction of D
o
cardiomyopathy through the compromised fractional shortening compared to baseline w
n
angiogenesis independently of STAT3, PGC1α, transgenic mice (40). These raise the possibility load
e
A K FTi,n aanlldy ,E wReK 1in/2v epsattihgwataeyds .m RNA expression tchaardt iaacc tivhaytpioenrt roopf hNy,C Xco1n tmraicgthilte bed yrsefluantecdti otno, d from
levels of angiogenesis-related genes in the heart. and postpartum heart failure in APJ-TG mice. http
The balance of pro-angiogenic and A previous work revealed that APJ deficient ://w
anti-angiogenic factors is important for proper mice exhibit resistance to pressure overload by ww
angiogenesis (35, 36). As shown in Fig. 5E, aortic banding (41). APJ-KO mice show blunted .jb
c
angiopoietin-1 (Angpt1), a pro-angiogenic factor myofiber hypertrophy after TAC. In addition, .org
(37), was induced by lactation in both WT and mechanical stretch induces cellular hypertrophy by/
APJ-TG mice, whereas the levels of Angpt1 and ANP expression in APJ expressing neonatal gu
e
s
mRNA were significantly decreased in APJ-TG rat cardiomyocytes (41). In our transgenic t o
n
mice compared with WT mice at non-pregnant model, overexpressed APJ may be activated by A
p
state and two weeks postpartum. Moreover, we mechanical stretch at basal condition, and may ril 2
found that thrombospondin-1 (Thbs1), an contribute to cardiac hypertrophy and , 2
0
endogenous inhibitor of angiogenesis (38, 39), dysfunction in APJ-TG mice. However, male 19
is significantly elevated in lactating APJ-TG and non-pregnant APJ-TG mice did not show
mice compared with non-lactating APJ-TG mice increase of ANP gene expression and myofiber
and lactating WT mice (Fig. 5E). These data hypertrophy. In addition, although ANP
suggest that the dysregulation of angiogenic expression was increased in the heart of
factor expression causes impaired angiogenesis lactating APJ-TG mice, myofiber hypertrophy
in lactating APJ-TG mice. was not observed compared with lactating WT
mice. These facts suggest that APJ activation by
Discussion mechanical stretch might not contribute to
While it has been demonstrated that the pathogenesis in APJ-TG mice.
apelin-APJ system is intimately related to It has been suggested that APJ receptor is
cardiac development, homeostasis, and diseases, involved in both Gαi- and Gαq-mediated
the effect of increased APJ expression in the signalings in the heart and 3T3-L1 cells (25, 42).
heart remains unclear. In the present study, we Cardiomyocyte-specific Gαq overexpressing
show that the cardiomyocyte-specific APJ mice die from heart failure in antepartum and
overexpression induces cardiac hypertrophy and postpartum periods, and show massive apoptosis
contractile dysfunction in mice. In addition, we in the heart at day one postpartum (16, 18).
found that lactation causes postpartum Since inhibition of cardiac apoptosis by caspase
8
inhibitor treatment or deletion of NIX gene, are associated with disorder of adaptive
which is a member of the Bcl2 family proteins, response to lactation in the heart. Because our
improves postpartum cardiac function in Gαq results suggest that postpartum cardiomyopathy
transgenic mice, apoptosis is thought to play a in APJ-TG mice is not involved in decreased
critical role in the development of peripartum STAT3 activity and elevation of cathepsin D
cardiomyopathy in this model (17, 18). On the expression, APJ-mice may be an effective tool
contrary, in our study, APJ-TG mice exhibited for further understanding the role of lactation in
increased apoptosis in the heart at four weeks postpartum cardiomyopathy. The details of
postpartum, but not at two weeks postpartum benefit and risk of lactation are poorly
(Fig. 4E). Furthermore, as compared with understood, however, our study may provide a
non-pregnant mice, antepartum APJ-TG mice new insight into the development of postpartum
did not show reduction of cardiac contractility cardiomyopathy associated with lactation.
(Fig. 3E). These differences in time point of In the present study, capillary density was
disease development between Gαq transgenic reduced in the heart of lactating APJ-TG mice,
and APJ-TG mice indicates that enhanced Gαq suggesting that the impaired myocardial
signaling and apoptosis may not be associated angiogenesis is related to the onset of
with onset of postpartum cardiomyopathy in lactation-induced postpartum cardiomyopathy in
APJ-TG mice. APJ-TG mice. Although previous studies have D
o
Lactation is thought to have beneficial revealed that apelin-APJ system in vascular wn
effects on cardiovascular systems (43, 44). cells is involved in angiogenesis (24, 46), our load
e
Fbruerathste-rfmeeodrien,g S aifsi rsrteeliant eedt atlo. (4im5)p rroevpeomrteedn t thoaft rceasrudlito msyuogcgyetsetss dtihsraut ptAs PthJ e oevxeprreexspsiroenss ipoant terinn d from
systolic function in peripartum cardiomyopathy of pro- and anti-angiogenic factors, such as http
patient. On the other hand, a previous work Angpt1 and Thbs1, resulting in deteriorated ://w
demonstrated that prolactin, which is an angiogenesis in the heart of lactating mice. It ww
important hormone for milk production, is has been reported that PGC1α is required for .jbc
cleaved by cathepsin D, and is converted into 16 expression of pro-angiogenic genes, including .org
kDa prolactin in the heart of Vegfa, Angpt1, Angpt2, Pdgfa, and Pdgfb in the by/
g
cardiomyocyte-specific STAT3 deficient mice heart (20), while PGC1α is normally expressed ue
s
(19). This cleaved prolactin inhibits cardiac in the heart of APJ-TG mice. As the Angpt1 t o
n
angiogenesis, resulting in postpartum gene is specifically decreased by the APJ A
p
cardiomyopathy (19). Thus, the effect of overexpression (Fig. 5E), it may impair ril 2
lactation on peripartum cardiomyopathy remains myocardial angiogenesis independently of , 2
0
1
controversial. In our study, non-lactating PGC1α. Further study is needed to determine 9
APJ-TG mice did not develop postpartum how APJ overexpressed in cardiomyocytes
cardiomyopathy. This result has clearly shown affects expression of Angpt1 and Thbs1 genes.
that lactation is critical for onset of postpartum We demonstrated that APJ overexpression in
cardiomyopathy in APJ-TG mice. In addition, cardiomyocytes contributes to postpartum heart
we previously demonstrated that lactation failure in mice, whereas cardiac expression level
causes cardiac contractile dysfunction in of APJ in the patients with postpartum
pregnancy-associated hypertensive (PAH) mice cardiomyopathy is unknown. Interestingly, a
in the postpartum period (7). PAH mice exhibit functional SNP has been identified in the
concentric cardiac hypertrophy and marked 5’-flanking region of APJ gene in Japanese
fibrosis by hypertension during late pregnancy, patients of brain infarction (47). This SNP
while cardiac contractility is preserved (7). affects DNA binding of Sp1 transcription factor
Although the genetic background and and modulates APJ gene expression levels (47).
antepartum maternal cardiac phenotypes of It would be important to reveal the association
APJ-TG mice and PAH mice were different, between postpartum cardiomyopathy and gene
both mice show reduction of cardiac mutations that increases APJ expression.
contractility by lactation. This indicates that In our study, non-pregnant APJ-TG mice
multiple qualitative changes in antepartum heart showed cardiac contractile dysfunction,
9
eccentric hypertrophy, and increased BNP gene defects. However, in patients with peripartum
expression, while features of heart failure, such cardiomyopathy, little is known about cardiac
as abnormal breathing and pleural effusion, function and BNP levels before onset of
were not observed. This suggests a possibility peripartum cardiomyopathy, because women
that these pre-existing non-severe cardiac who have no symptoms of heart failure lack the
phenotypes influence development of opportunity to examine their cardiac parameters.
lactation-induced postpartum cardiomyopathy in To investigate the relationship between
APJ-TG mice. Although one of the definitions pre-existing non-severe cardiac defects and
of peripartum cardiomyopathy is without history onset of peripartum cardiomyopathy might be
of heart disease (48), non-pregnant women, who important for prediction of peripartum
are going to develop peripartum cardiomyopathy.
cardiomyopathy, might have non-severe cardiac

Acknowledgement
We are grateful to Dr. Ryo Takeda for establishing transgenic mice and to Dr. Yasunobu Hirata
for helpful discussion. This study was supported by Grant-in-Aid for Scientific Research B (to J.I.,
Grant Number 23300152) from the Ministry of Education, Culture, Sports, Science, and Technology
of Japan. D
o
w
n
Conflict of interest loa
d
e
The authors declare that they have no conflicts of interest with the contents of this article. d
from
Author contributions http
K. M., J. I. and A. F. designed research; K. M., C. S., T.I. and H. M. performed experiments and ://w
analyzed data; K. M. and A. F. wrote the manuscript; K. M. J. I., J. H. and A. F. discussed the results w
w
and commented on the manuscript. .jb
c
.org
References b/
y
1. Li, J., Umar, S., Amjedi, M., Iorga, A., Sharma, S., Nadadur, R. D., Regitz-Zagrosek, V., and gu
e
s
Eghbali, M. (2012) New frontiers in heart hypertrophy during pregnancy. Am. J. Cardiovasc. t o
n
Dis. 2, 192-207 A
p
2. Chung, E., and Leinwand, L. A. (2014) Pregnancy as a cardiac stress model. Cardiovasc. Res. ril 2
101, 561-570 , 2
0
3. Mesa, A., Jessurun, C., Hernandez, A., Adam, K., Brown, D., Vaughn, W. K., and Wilansky, S. 19
(1999) Left ventricular diastolic function in normal human pregnancy. Circulation 99, 511-517
4. Hanwell, A., and Linzell, J. L. (1973) The time course of cardiovascular changes in lactation in
the rat. J. Physiol. 233, 93-109
5. Sakanashi, T. M., Brigham, H. E., and Rasmussen, K. M. (1987) Effect of dietary restriction
during lactation on cardiac output, organ blood flow and organ weights of rats. J. Nutr. 117,
1469-1474
6. Wang, X., Hole, D. G., Da Costa, T. H., and Evans, R. D. (1998) Alterations in myocardial lipid
metabolism during lactation in the rat. Am. J. Physiol. 275, E265-E271
7. Murata, K., Saito, C., Ishida, J., Hamada, J., Sugiyama, F., Yagami, K., and Fukamizu, A.
(2013) Effect of lactation on postpartum cardiac function of pregnancy-associated hypertensive
mice. Endocrinology 154, 597-602
8. Sliwa, K., Fett, J., and Elkayam, U. (2006) Peripartum cardiomyopathy. The Lancet. 368,
687-693
9. Hilfiker-Kleiner, D., Haghikia, A., Nonhoff, J., and Bauersachs, J. (2015) Peripartum
cardiomyopathy: current management and future perspectives. Eur. Heart J. 36, 1090-1097
10. Ntusi, N. B., and Mayosi, B. M. (2009) Aetiology and risk factors of peripartum
cardiomyopathy: a systematic review. Int. J. Cardiol. 131, 168-179
10