Transgenic myocardial overexpression of prokineticin receptor-2 ...

5 downloads 0 Views 2MB Size Report
Transgenic myocardial overexpression of prokineticin receptor-2 (GPR73b) induces hypertrophy and capillary vessel leakage. Kyoji Urayama1, Deniz B.
Cardiovascular Research (2009) 81, 28–37 doi:10.1093/cvr/cvn251

Transgenic myocardial overexpression of prokineticin receptor-2 (GPR73b) induces hypertrophy and capillary vessel leakage ´lia Guilini1, Stefan Frantz2, Georg Ertl2, Nadia Messaddeq3, Kyoji Urayama1, Deniz B. Dedeoglu1, Ce 1 and Canan G. Nebigil * 1

´rieure de Biotechnologie de Strasbourg, Bld. Se ´bastien Brandt BP. 10413, F-67412 CNRS/ULP, UMR 7175-LC1, Ecole Supe Illkirch, France; 2Medizinsche Klinik und Poliklinik I, Herz-/Kreislaufzentrum, University Wurzburg, Wurzburg, Germany; and 3 ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, CNRS/ULP UMR 7104, INSERM U596, BP. 10142, F-67404 Institut de Ge Illkirch, France Received 24 January 2008; revised 22 August 2008; accepted 12 September 2008; online publish-ahead-of-print 20 September 2008 Time for primary review: 33 days

KEYWORDS

Aims Prokineticins are small secreted bioactive molecules. They exert their biological activity by binding to two G protein-coupled receptors. Previously, we have shown that the overexpression of prokineticin receptor-1 (PKR1) in transgenic (TG) mouse hearts induced neovascularization. Since PKR1 and PKR2 are 85% identical and expressed in cardiovascular tissues, we hypothesized that PKR2 may also contribute to cardiomyocyte growth and vascularization. Methods and results We have generated TG mice overexpressing PKR2 in cardiomyocytes. TG mice exhibit increased hypertrophic gene expression and heart-to-body weight ratio accompanied by an increased length of cardiomyocytes at the age of 12 weeks. Increased left ventricular end-systolic and diastolic diameters without cardiac dysfunction at the age of 24 weeks indicate that TG mice have an eccentric hypertrophy with compensated cardiac function. Quantitative morphological analysis showed that TG hearts have a normal microvessel density and number of branch points. However, they exhibit increased abnormal endothelial cell shape and ultrastructure, changed cellular distribution of a tight junction protein zona occludens-1 (ZO-1), and vascular leakage in heart without a rise of angiogenic factor levels at early and late age. The application of media conditioned by H9c2 cardioblast cells overexpressing PKR2 significantly induced impaired ZO-1 localization in H5V endothelial cells, mimicking the TG model. Conclusion These findings provide the first genetic evidence that cardiomyocyte PKR2 signalling leads to eccentric hypertrophy in an autocrine regulation and impaired endothelial integrity in a paracrine regulation without inducing angiogenesis. These TG mice may provide a new genetic model for heart diseases.

1. Introduction Prokineticins, comprising prokineticin-1 (also called EG-VEGF) and prokineticin-2 (also called Bv8), are secreted bioactive proteins.1,2 Prokineticins are involved in regulating diverse biological processes that include angiogenesis,3 haematopoiesis,4 monocyte differentiation,5 macrophage activation,6 and neuronal survival7 olfactory bulb activation,8,9 gastrointestinal motility,2 pain sensitization,10,11 circadian rhythm,12,13 coordination of circadian behaviour, and physiology.14 Prokineticins exert their biological activities by stimulating two closely related receptors: PKR1 and PKR2.15,16 Prokineticin-2 is the most potent agonist for both receptors

* Corresponding author. Tel: þ33 390 24 47 56; fax: þ33 390 24 48 29. E-mail address: [email protected]

under physiological condition.16,17 Both receptors mainly use the Gq signalling pathway and are ubiquitously expressed in mammalian tissues.18–20 It was shown that Gq/G11 signalling is an essential pathway to regulate cardiac development and hypertrophy.21 Some other Gq-coupled receptors have been shown to be involved in the development of hypertrophy and the protection of cardiomyocytes.22–27 Moreover, Gq/ G13 contributes to vessel formation.28 However, the role of prokineticin receptors in cardiovascular function has not yet been elucidated until recently. We have shown that the activation of PKR1 in coronary endothelial cells induces vessel-like formation. PKR1 signalling protects cardiomyocytes against hypoxia. Moreover, transient PKR1 gene transfer reduces mortality and preserves left ventricular (LV) function by promoting angiogenesis and cardiomyocyte survival in the

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008. For permissions please email: [email protected].

Downloaded from by guest on December 21, 2015

GPCR; Prokineticin; Fenestration; Transgenic mice; Cardiomyocyte; ZO-1

29

Fenestration and cardiac PKR2 signalling

coronary ligation, a mouse model for myocardial infarction.29 We also showed that the overexpression of PKR1 in transgenic (TG) mice heart displayed no spontaneous abnormalities in cardiomyocytes, but increased capillary density and number of coronary arterioles. PKR1 signalling upregulates its own ligand, prokineticin-2 as a paracrine factor to induce the proliferation and differentiation of the epicardial-derived progenitor cells, thereby regulating post-natal coronary angiogenesis and vasculogenesis.20 Since PKR1 and PKR2 are 85% identical and expressed in cardiovascular tissues, we hypothesize that PKR2 may also contribute to cardiomyocyte growth and vascularization. In this study, we investigate the role of PKR2 in the heart by generating TG mice overexpressing PKR2 in cardiomyocytes. We found that PKR2, unlikely PKR1, induces hypertrophy in cardiomyocyte and vascular leakage in a paracrine fashion. Our results provide novel insight into the functional significance of cardiac PKR2 signalling on vascular integrity by a paracrine regulation.

using the RNase-Free DNase Set. Semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) was performed on 0.5–5 mg of total RNA extracted from littermate non-TG (NTG) and TG mice, using the GAPDH as an internal control as described previously.20 The primers used are shown in Table 1. All PCR-derived fragments were sequenced to confirm their predicted composition. All values given are representative of at least three duplicated independent experiments obtained from three to four different heart samples. Real-time quantitative polymerase chain reaction (qPCR) was performed with MyiQ cycler (Bio-Rad) using SYBR Green system (Bio-Rad). All data of threshold Cycle (Ct) were normalized by Ct of house-keeping gene 36B4.

2. Methods

2.4 Immunostaining analyses

2.1 Generation of transgenic mice

Immunofluorescence was performed on 5 or 10 mm heart cryosections or H5V cells as described previously. The cryosections or cells were fixed with 3.7% formaldehyde and permeabilized by 0.5% Triton X-100. The following antibodies were used: a polyclonal antibody against human PKR2 (GPR73B, Abcam), MF-20 (Hybridoma bank) a polyclonal antibody against the endothelial marker PECAM-1 (Santa Cruz), a monoclonal antibody against vascular smooth muscle alpha-actin (Sigma Chemical Co.), anti-zona occludens-1 (ZO-1) (Invitrogen), and anti-mouse CD68 (Serotec). Nuclei were stained with DAPI. All values given are representative of at least three independent experiments obtained from three to four different heart samples. The measurements of immunofluorescence intensity were performed with a Leica TCSNT confocal microscope or fluorescent microscope. Signal intensity was quantified on digitalized images and calculated as the product of averaged pixel intensity per high-power field or per cell number. Some of the fluorescent signals were obtained with confocal microscope and images were controlled by LEICA software. The laser was chosen according to the sample and compared pixel intensity and pixel distribution. These data were analysed using the pixel data from which background intensity was subtracted.20

2.2 Analysis of gene expression by reverse transcriptase–polymerase chain reaction and real-time quantitative polymerase chain reaction Total RNA from mice hearts, H5V, and H9c2 cells were isolated using TRIZOLwReagent (Life Technologies) and treated with DNase

The hearts of NTG and TG mice (12- and 24-week-old male mice) were dissected, and the anatomy was assessed under the binocular microscope. The hearts were then cryopreserved and sectioned (5–10 mm) using standard techniques. Mallory tetrachrome staining was performed for histological analysis as described previously.27 Note that male and female TG mice exhibit same phenotype at 12 and 24 weeks (n ¼ 8).

2.5 Electron microscopy The hearts of NTG and both lines of TG mice were fixed by immersion in glutaraldehyde, post-fixed with osmium tetroxide, and embedded in epoxy resin using routine methods. Thin sections were post-stained with uranyl acetate and Reynolds lead citrate and photographed with a Philips CM10 electron microscope. The quantification of caveolae-like structure was performed by counting caveolae-like structure per micrometer of vessel perimeter on electron micrographs at 30 000 magnification; 20 microvessels analysed for each genotype.20

2.6 Echocardiography Echocardiographic studies were performed under light anaesthesia with spontaneous respiration using ketamine (100 mg/kg) for 24-week-old male mice.30 An ultrasonographer experienced in rodent imaging and blinded to the mouse genotype performed the echocardiography, operating a Toshiba Aplio and a 15 MHz transducer. Short-axis two-dimensional echocardiographic images were obtained at the mid-papillary and apical levels of the left ventricle and stored as digital loops. Frame acquisition rates using the loop mode reached 100 MHz, allowing excellent temporal resolution for

Downloaded from by guest on December 21, 2015

We generated TG mice overexpressing full-length human PKR2 under the control of the myosin heavy chain (a-MHC) promoter that were previously shown to specifically direct gene expression to cardiomyocytes. A plasmid containing 5.5 kb from a-MHC gene (including the promoter and the first three untranslated exons) was used.20,24 A 1.2 kb XhoI–KpnI restriction fragment of human PKR2 cDNA containing the whole open-reading frame was then subcloned into the XhoI site of the a-MHC promoter plasmid. The recombinant plasmid (p-MHC-PKR2) was linearized, microinjected into pronuclei of fertilized CD1 mouse eggs which were reimplanted into pseudo-pregnant C57BL/B6 foster mothers (Mouse Clinic Institute, Illkirch) as described before.20 Southern blot of tail DNA hybridized with the PKR2 cDNA labelled with alpha[32P] dATP was used to prove the presence of the transgene in founder mice. A total of 10 mg of BamHI-restricted genomic DNA from WT mice or mice from lines 1 or 25 were analysed by Southern blotting with probe A (NdeI–XhoI) from the a-MHC gene, and probe B (XhoI–EcoRV) containing hPKR2 coding sequences. Copy number was estimated by comparing the intensities (determined by PhosphorImager; Fuji) of the transgene fragment (probe A, XhoI–NdeI) with that of the 6 kb fragment corresponding to the endogenous a-MHC locus. Further genotyping was performed by polymerase chain reaction (PCR) analysis of tail DNA using forward and reverse primers located in the a-MHC and PKR2 sequences, respectively (Table 1). A 296 bp fragment was thus amplified in TG founders. Most of the experiments described here were performed with mice from line 1. Thereafter the transgene was maintained in the hemizygous state by breading male TG animals with B6D2F1 females (The Jackson Laboratory). TG mice were recovered at Mendelian frequency, and survived to adulthood. No sudden death was observed. All the experiments were carried out using F2-F4 generation male mice. All animal experimentation was performed in accordance with institutional guidelines of the French Animal Care Committee, with the European regulationapproved protocols and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Anatomy and histomorphological analysis

30

K. Urayama et al.

Table 1 Oligos for reverse transcriptase–polymerase chain reaction experiments and genotyping Sequence (50 -30 )

Gene Forward Reverse

CTGCTGGAGAGGTTATTCCTCG GGAAGAGTGAGCGGCGCATCAAGG

Mouse b-MHC

Forward Reverse

ACAGAGGAAGACAGGAAGAAC TGCAAAGGCTCCAGGTCTGAGGGC

Mouse ANF

Forward Reverse

ATGGGCTCCTTCTCCATCAC TCTTCGGTACCGGAAGCT

Mouse GATA4

Forward Reverse

CCCTCCCTCCAGTGGTGCCTC TTGGAGCTGGCCTGCGATGTC

Mouse PKR1

Forward Reverse

TCTGGGTGAGAATACCACAG GAGACGGTACGCAAGTAGTT

Mouse PKR2

Forward Reverse

GAAACACTAGCTTTGCAC GCAGAGCGTTGGTGGAGAC

Mouse GAPDH

Forward Reverse

TGAGGCCGGTGCTGAGTATGTCG CCACAGTCTTCTGGGTGGCAGTG

Genotyping for TG mice

Forward Reverse

CGGCACTCTTAGCAAACCTCAGGCACCCTTA GTGAAACTGGTGTTTCCATTCTGGC

Rat PKR1

Forward Reverse

GCCATTCACTTTCAGCTAT TGCAGCCGTTTGACACTTC

Rat PKR2

Forward Reverse

TCCACGTGGGCCTATCTCCC CTCCCAGGAAAGCTGACGTAC

Rat GAPDH

Forward Reverse

ATGGGAAGCTGGTCATCAAC CCACAGTCTTCTGAGTGGCA

Human PKR2

Forward Reverse

ATATCTCGCCATCGTTCACC AGCACATAGGCCGTGAGAATGC

Mouse 36B4 (real-time)

Forward Reverse

AGATTCGGGATATGCTGTTGG AAAGCCTGGAAGAAGGAGGTC

Mouse a-MHC (real-time)

Forward Reverse

GAGATTTCTCCAACCCAG TCTGACTTTCGGAGGTACT

Mouse b-MHC (real-time)

Forward Reverse

CTACAGGCCTGGGCTTACCT TCTCCTTCTCAGACTTCCGC

Mouse ANF (real-time)

Forward Reverse

CCTGTGTACAGTGCGGTGTC CCTGCTTCCTCAGTCTGCTC

Mouse GATA4 (real-time)

Forward Reverse

TCTCACTATGGGCACAGCAG GCGATGTCTGAGTGACAGGA

two-dimensional analysis. At the same anatomic levels, short-axis M-mode images were obtained with a sweep speed of 100 mm/s. Endocardial borders were traced at end-systole and end-diastole utilizing a prototype off-line analysis system (NICE, Toshiba Medical Systems, The Netherlands) as described recently.30 Using the end-systolic and -diastolic areas, fractional area changes were calculated at both levels as [(end-diastolic area 2 end-systolic area) / end-diastolic area].

2.7 Haemodynamic assessment Cardiac haemodynamic analyses were assessed under light anaesthesia with spontaneous respiration using isoflurane (initially 5% and continuously 1.5%) as described recently for 12-week-old male mice (NTG and TG), and ketamine (100 mg/kg) for 24-week-old male mice (NTG and TG).29 Body temperature was maintained at 378C. A 1.4-French high-fidelity Millar pressure catheter (Millar Instruments, Houston, TX, USA) was inserted via the right carotid

artery into the left ventricle. After LV function and heart rate had stabilized, LV systolic pressure, end-diastolic pressure, developed pressure, maximal positive (dP/dt) or negative (2dP/dt) were recorded. The calibration of the Millar catheter was verified before and after each measurement.

2.8 Evans blue permeability assay One hundred and fifty microlitres of a 1% Evans blue dye (Sigma-Aldrich) solution in saline was injected into the tail vein of non-anaesthetized TG and NTG control mice. The appearance of blue spots was monitored 30 min after injection.31

2.9 Tissue lyses and immunoblotting Mouse hearts were homogenized in lysate buffer (50 mM Tris–HCl, pH 6.8, 1 mM EDTA, pH 8.0, 1% NP-40, 1 mM Na3VO4, 0.1% SDS, 100 mM NaCl) and protease inhibitor cocktail (Roche) and then centrifuged

Downloaded from by guest on December 21, 2015

Mouse a-MHC

31

Fenestration and cardiac PKR2 signalling and collected the lysate.24 The proteins were separated on 7–10% SDS–PAGE and blotted to PVDF membranes. Antibody/antigen complexes for phosphorylated-ERK1/2 (Cell Signaling) and ZO-1 were detected with an enhanced chemiluminescence kit (Pierce) as per the manufacturer’s instructions. Loading homogeneity was verified by stripping and reprobing the blots with total ERK and GADPH antibody using the enhanced chemiluminescence kit, according to the manufacturer’s recommendations. All values given are representative of at least three duplicated independent experiments.

2.10 Cell culture The H9c2 cardioblast cell line derived from embryonic rat heart was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM supplemented with 10% FCS, penicillin G (100 U/mL), and streptomycin (100 mg/mL), under 5% CO2 at 378C. Cardiomyocytes from 12-week-old TG and NTG mice hearts (n ¼ 3) were isolated, the size were calculated or treated with prokineticin-2 for indicated time, and cell lysates were utilized for western blot analyses as described previously.24 H5V endothelial cells derived from mouse heart were a kind gift of Dr Annunciata Vecchi (Istituto Clinico Humanitas, Rozzano, Italy). H5V cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, penicillin G (100 U/mL), streptomycin (100 mg/mL), and glutamine (1 mM) under 5% CO2 at 378C.29

3. Results 3.1 Characterization of PKR2 expression in cardiovascular tissues and generation of transgenic mice Quantitative analysis on RNA extracts revealed that both PKR1 and PKR2 were expressed in the H9c2 cardioblast cell line derived from rat heart as well as in H5V capillary endothelial cells derived from mouse heart, and rat testis (Figure 1A). Both receptors also exist in mouse heart (Figure 1C). These data show that PKR2 is also post-natally expressed in cardiac cells and tissues might be functional in the heart. To determine a potential role for the PKR2 and a possible action in heart, we generated TG mice overexpressing human PKR2 in cardiomyocytes (Figure 1B, left). Two TG founders (number 1 and 25) were identified estimating copy numbers approximately 12 and 10 in lines 1 and 25,

Downloaded from by guest on December 21, 2015

2.11 Culture H5V cells with media conditioned by transfected H9c2 cells H9c2 cells (104 cells/well) grown onto eight-well glass chambers were transfected with control vector, pcDNA3.1 or plasmid PKR2 cDNA, using lipofectamine 2000 (Invitrogen). After 24 h transfection, the medium was replaced with a fresh culture medium to remove transfection complex. After further 24 h incubation at 378C under 5%CO2, the medium was collected from transfected H9c2 cells. The serumstarved (2% serum) H5V cells cultured onto eight-well glass chamber were treated with the medium conditioned by H9c2 cells overexpressing pPKR2 or control vector plasmid for 1, 4, and 24 h at 378C under 5%CO2, H5V cells were fixed, and immunostaining for ZO-1 was performed. All values given are representative of at least three duplicated independent experiments.

2.12 Angiogenesis antibody array The angiogenesis antibody array was performed according to the protocol (RayBio Mouse Angiogenesis Antibody Array, RayBiotech, Inc.) on 250 mg of total protein from mouse heart. First, membranes were blocked with blocking buffer for 30 min at room temperature and then the blocked membranes were incubated with 250 mg of total protein from mouse heart for overnight at 48C. After washing the membrane with two different wash buffers, the membranes were incubated with biotinylated antibody for overnight at 48C and then the membranes were incubated with HRP-conjugated streptavidin for 2 h at room temperature. The signals were detected on X-ray film. Each signal density was quantified and normalized with positive and negative controls. All values given are representative of at least two duplicated independent experiments.

2.13 Statistical analysis The Student t-test or ANOVA was used to examine the differences between experimental groups. All data were presented as mean + SE. Quantitative analyses were performed with the evaluators blinded to genotype. The chosen significance criterion was P , 0.05.

Figure 1 (A) Reverse transcriptase–polymerase chain reaction analyses for prokineticin receptors and house-keeping gene GAPDH on RNA extracts. PKR1 and PKR2 expressions were found only in the presence of reverse transcriptase (þRT), indicating no genomic DNA contamination in RNA extracts. (B) Schematic representation of the transgene construct (not at scale). Black boxes indicate exons 1–3 of the a-myosin heavy chain (a-MHC) gene, which correspond to 50 -untranslated sequences (left). Genomic DNA from WT and transgenic (TG) lines 1 or 25 were analysed by Southern blotting with probe A (NdeI–XhoI) from the a-myosin heavy chain gene (middle), and probe B (XhoI–EcoRV) containing hPKR2 coding sequences, as indicated (right). (C ) The reverse transcriptase-polymerase chain reaction analysis of transgene hPKR2 in hearts of both transgenic lines (upper panel) and endogenous prokineticin receptors in TG#1 heart (lower panel) and testis samples (right panel). (D) Immunostaining analysis on cryosectioned hearts revealed that PKR2 expression (red) is localized in cardiomyocyte (MF-20-green) in non-transgenic (NTG) hearts and transgene did not alter the localization of PKR2 (upper). Confocal analyses revealed increase level of PKR2 in the membrane of isolated transgenic cardiomyocytes. (E) Histogram shows relative PKR2 protein levels (density/pixel) in transgenic vs. nontransgenic hearts or isolated cardiomyocytes—Epi (sub and epicardium), Myo (myocardium), Endo (endocardium), Cmyo (cardiomyocytes). (F ) Prokineticin-2 induces ERK1/2 phosphorylation/activation within 15 min in non-transgenic and transgenic cardiomyocytes by 1.4-fold and 2.7-fold, respectively.

32

K. Urayama et al.

respectively (Figure 1B, middle). Southern analysis (Figure 1B, middle) with hPKR2-derived probe B revealed the presence of DNA fragments of expected sizes only in TG lines. RT–PCR analyses confirmed PKR2 expression in both TG hearts (Figure 1C, upper). RT–PCR analyses on heart RNA samples (TG#1) showed that the targeted expression of the transgene is expressed in the heart without altering endogenous expression of two related receptors in heart (Figure 1C, lower). No transgene expression was observed in non-cardiac tissues such as testis (Figure 1C). Co-staining of cryosectioned heart samples with PKR2 and cardiomyocytespecific MHC (MF-20) revealed the presence of PKR2 protein in NTG sibling cardiomyocytes and increased level of PKR2 in TG cardiomyocytes (Figure 1D), as confirmed in the TG-isolated cardiomyocytes. Confocal microscopic analyses on the heart sections and isolated cardiomyocytes revealed a significant increase in PKR2 expression levels, especially in the TG hearts, in comparison with NTG mice (subepicardium, 1.7-fold; myocardium 3.2-fold; endocardium, 2.2-fold; isolated cardiomyocytes 4.7-fold) (Figure 1E, histogram). Moreover, prokineticin-2 induces the phosphorylation of MAPK in NTG and TG-isolated cardiomyocytes by 1.4- and 2.7-fold, respectively, indicating an augmentation of PKR2 signalling in TG cardiomyocytes.

We next investigated whether genetically overexpressed PKR2 in mouse cardiomyocytes induces any cardiac pathology as observed in TG mice overexpressing other G protein coupled receptors (GPCR).32 The size of the ventricular diameter and heart-to-body weight ratio were significantly increased in 12-week-old TG hearts, which were not significantly altered in 24-week-old TG hearts (Figure 2A). Echocardiographic analyses confirmed that the LV end-diastolic and -systolic diameters (LVEDDs and LVESDs) were significantly increased in 24-week-old TG hearts at the expense of lack of significant differences in resting LV morphology and function (Figure 2B, Table 2). As shown in Table 2, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between NTG and TG mice at 24 weeks. Haemodynamic analyses on the 12- and 24-week-old TG mice revealed no abnormal cardiac functions compared with their sibling NTG mice (Table 3). Isolated cardiomyocytes from 12-week-old TG hearts had a significant increase in length by 23% (Figure 2C). RT–PCR analysis of 12- and 24-week-old TG heart mRNA demonstrated an increase in a- and b-MHC, ANF, and GATA-4 gene expressions (Figure 2D, and histogram, P , 0.05). These changes in the hypertrophic marker expression demonstrate the activation of a molecular programme for cardiac hypertrophy in both 12- and 24-week-old TG hearts.

3.3 Heart ultrastructural analysis In semi-thin sections of TG hearts (12 weeks), an increased glycogen deposition and enlarged blood vessels were observed in comparison with NTG hearts (Figure 3A, upper and lower panels). Electron microscopy revealed that cardiomyocytes in TG mice have normal nuclei, intact sarcolemma. Notably, no evidence for myocardial apoptosis or necrosis but significant inflammatory cell infiltrates around the coronary vessels was found in the TG hearts. TG cardiomyocytes exhibit abnormal mitochondrial proliferation

Downloaded from by guest on December 21, 2015

3.2 Heart morphology and cardiac functions

Figure 2 (A) Histological analysis with Mallory tetrachrome staining on heart sections (n ¼ 8). Histogram shows heart-to-body weight ratios. (B) Echocardiographic analysis in hearts revealed significant increase in left ventricular end-diastolic diameter (LVEDD) and significant increase in end-systolic diameter (LVESD) in 24-week-old transgenic (TG) hearts (n ¼ 6, P , 0.05). (C ) Isolated cardiomyocytes size in transgenic and non-transgenic (NTG) hearts (n ¼ 3, 12-week-old). (D) Reverse transcriptase–polymerase chain reaction analysis of hypertrophic markers on RNA extracts from transgenic and non-transgenic hearts normalized by house-keeping gene GAPDH. Real-time quantitative polymerase chain reaction analyses (histogram) for transcripts of hypertrophic markers in 24-week-old transgenic hearts. *P , 0.05.

with densely packed cristae, and T-tubules enlargement accompanied with increased number of serial assembly of sarcomeres (Figure 3B). Note that similar histopathology was observed in both TG lines.

3.4 Coronary capillary network and arterioles of transgenic heart To investigate the impact of cardiac-PKR2 signalling on potential angiogenic activity, blood vessels were identified by immunostaining of tissue sections for PECAM-1, an endothelial specific marker. The density of PECAM-1-positive vessels was not altered in TG hearts at any ages [Figure 4A (upper panel) and B]. By staining serial heart sections with

33

Fenestration and cardiac PKR2 signalling

3.6 Paracrine effects of cardiomyocyte-PKR2 signalling on endothelial cell junction

Table 2 Echocardiographic analyses Genotype

NTG (24 weeks)

TG-PKR2 (24 weeks)

Heart rate (b.p.m.) LVEDD (cm) LVESD (cm) Posterior wall (cm) EF (%) M-mode FS (%)

566.9 + 13.92 0.317 + 0.008 0.193 + 0.007 0.086 + 0.003 65.07 + 2.13 41.7 + 2.12

574.3 + 13.52 0.349 + 0.007* 0.216 + 0.006* 0.085 + 0.004 70.64 + 4.93 42.0 + 2.29

Twenty-four-week-old mice were utilized (n ¼ 6 for each group). EDD, end-diastolic diameter; ESD, end-systolic diameter; EF, ejection fraction; FS, fractional shortening. *P , 0.05 shows significant differences in TG mice compared with NTG.

Downloaded from by guest on December 21, 2015

an antibody directed against the alpha-smooth muscle actin (a-SMA), which detects smooth muscle cells, no alteration was detected in smooth-muscle-coated vessel numbers in TG hearts compared with those of NTG littermates [Figure 4A (lower panel) and B]. Accordingly, none of the known angiogenic factor levels were altered in TG hearts compared with NTG hearts (Figure 4C). In contrast, the diameters of small blood vessels in the TG hearts were strongly enlarged (Figure 4A, green). The mean blood vessel diameter was increased by 25% (P , 0.001) in the PKR2-TG hearts compared with NTG, but was highly variable (Figure 4A). Figure 4D shows that CD68-positive monocytes and macrophages are extravasated across microvessels in TG hearts, indicating hyperpermeability of cardiac vessels.

To further analyse the basis for vascular leakage in TG hearts, we performed a detailed analysis of tight junction proteins in cardiac endothelial cells by immunolabelling and confocal laser scanning microscopy. Analysis of the tight junction protein, ZO-1, revealed reduced junctional localization of staining intensity in TG hearts. The microaneurysms typical for the PKR2-TG hearts exhibited highly abnormal staining for ZO-1 proteins (Figure 6A). Co-staining with PECAM-1 revealed that ZO-1 is regularly expressed between endothelial cells on the tight junctions in NTG cardiac vessels. However, not all the tight junctions of endothelial cells have ZO-1 protein in TG vessels. Western blot analysis on heart lysate revealed significantly decreased level of ZO-1 in TG hearts compared with NTG hearts (Figure 6B). In addition, conditioned media of H9c2 cardioblast cells overexpressing PKR2 induces disrupted ZO-1 distributions

3.5 Vascular, permeability, leakage, and ultrastructure The invaginated plasma membrane with labyrinth-like structures, interconnected endothelial cells, and clusters of vacuoles were identified in the vessels of 12-week-old TG hearts (Figure 5A, upper panel). Fenestrae, a small plasma membrane discontinuity of the endothelial cells in the particular capillary beds, were found in the TG hearts. The endothelial lining of 30% of these vessels showed segmental thinning, was frequently interrupted, and became fenestrated in TG hearts. Figure 5A (lower panel) shows a 3-fold increase in the number of caveolae-like structures. Consistent with an increase of fluid transport, many TG vessels exhibited prominent swelling, which was not seen in NTG. After injection of Evans blue dye perfusion and fixation, 24-week-old TG hearts appeared visibly bluer than that of NTG littermates, indicating a possible increase in vascular leakage in TG hearts (Figure 5B). Note that similar fenestration and vessel leakage were observed at both 12- and 24-week-old TG heart vessels.

Figure 3 (A) Semi-thin sections demonstrated increased glycogen depositions in transgenic (TG) hearts (upper panel) and vessel size (lower panel). Arrows indicate small dots, which correspond to glycogen deposition. (B) Electron microscopic analyses revealed increased serial sarcomere numbers and mitochondrial proliferation with normal gap junctions in transgenic hearts. m, mitochondria; z, Z bands; gp, gap junctions.

Table 3 Haemodynamic analyses (n ¼ 6 for each group) Genotype

NTG (12 weeks)

TG-PKR2 (12 weeks)

NTG (24 weeks)

TG-PKR2 (24 weeks)

Heart rate (b.p.m.) Maximum pressure (mmHg) End-diastolic pressure (mmHg) Maximum dP/dt (mmHg/s) Minimum dP/dt (mmHg/s)

438.4 + 33.7 89.56 + 3.59 212.14 + 2.44 8745.6 + 718.4 25404.4 + 502.1

474.7 + 20.5 95.95 + 6.95 211.57 + 3.27 9113.9 + 944.9 26546.3 + 339.7

512.2 + 14.0 101.7 + 1.93 0.45 + 1.03 8839.7 + 395.1 27803.3 + 363.1

512.1 + 18.3 95.3 + 3.68 20.23 + 0.64 8696.2 + 321.8 27188.3 + 451.8

34

K. Urayama et al.

in coronary endothelial, H5V cells (Figure 6C), indicating a paracrine regulation of endothelial cell structure by cardiomyocyte-PKR2 signalling.

4. Discussion

Figure 5 (A) Electron microscopic analysis of the heart of non-transgenic (NTG) and transgenic (TG) mice revealed that plasma membranes were invaginated, and labyrinth-like structures of the endothelial cells were observed in the transgenic blood vessels (upper panel). Interconnected endothelial cells and increased caveolae-like structures were identified in the transgenic vessels (lower panel). (B) Evans blue staining revealed vascular leakage in transgenic mice heart. Arrow indicates haemorrhage-like staining in the transgenic hearts.

The major findings in this study are that PKR2 signalling pathways in TG cardiomyocytes induced eccentric hypertrophy and increased capillary endothelial permeability by an autocrine and paracrine regulation, respectively (Figure 6D). In this study, we first investigated the pathological consequences of genetically overexpressed novel GPCR, PKR2 (GPR73b) in the heart. Interestingly, we also found that the ratio of heart to body weight and hypertrophic marker expression were slightly increased in 12-week-old TG mice, even though functional cardiac indexes remained unchanged. This suggested that foetal gene re-expression may be a more sensitive indicator for cardiac hypertrophy. Despite increased hypertrophic markers and LV chamber dimension in 24-week-old TG hearts, shortening fraction and ejection fraction (indicators of LV contractility) and cardiac output did not differ between NTG and TG mice, which indicates that LV function in older TG mice is well compensated. Indeed, we found that the length of cardiomyocytes was increased in TG mice, suggesting that cardiomyocyte elongation upon excessive PKR2 signalling may contribute to the eccentric hypertrophy in TG mice. This type of hypertrophy was accompanied by the induction of embryonic gene expression, the lack of significant differences in resting LV morphology and function, with increased LV end-systolic or -diastolic dimension consisting of human eccentric hypertrophy.33 Note that this phenotype is associated with increased cardiomyocytes in length,

Downloaded from by guest on December 21, 2015

Figure 4 (A) Representative illustration of capillary density by an endothelial marker, PECAM-1 (red) (n ¼ 4), and arteriole numbers assessed by a smooth muscle-specific marker, a-smooth muscle-specific actin (a-SMA) staining (green) (n ¼ 4) on cryosectioned hearts. Original magnification: 11 and 20, respectively. Arrow shows increased diameter of arteriole with irregular smooth muscle surroundings in transgenic (TG) heart. (B) Quantitative analyses of number of PECAM-1þ capillary and a-SMAþ arterioles. (C ) Angiogenic antibody arrays (RayBio Mouse Angiogenesis Antibody Array, RayBiotech, Inc.) on heart samples obtained from non-transgenic (NTG) and transgenic hearts (duplicated experiments n ¼ 3). None of the angiogenic factor levels was increased in transgenic hearts. (D) Representative illustration of a-smooth muscle-specific actin and macrophage-specific marker CD68 on cryosectioned non-transgenic and transgenic hearts (n ¼ 4). CD68 (red)-positive macrophages were extravasated across microvessels in transgenic hearts.

Fenestration and cardiac PKR2 signalling

35

increased sarcomere numbers as observed in TG mice overexpressing MEK5-ERK5 in heart.34 In TG mice, cardiacspecific overexpression of constitutively active delta-PKC and epsilon-PKC isoforms has been shown to cause identical non-pathological cardiac hypertrophy.35,36 TG mice with cardiac-restricted expression of an activated MEK1 exhibit concentric hypertrophy without signs of cardiomyopathy or lethality.37 Therefore, overexpression of signalling intermediates downstream of PKR2 produces phenotypes that are remarkably similar to that observed in mice overexpressing PKR2 on cardiomyocytes. Further studies are required to determine the role of the Gq, MEK5-ERK5, and MEK1-ERK1/2 signalling pathways in PKR2-dependent eccentric hypertrophy. We have shown previously that PKR1 signalling stimulates cardiomyocyte survival pathway to protect cardiomyocytes against hypoxic insult.29 From our data presented here, it is clear that PKR1 is involved in survival signalling, whereas PKR2 in hypertrophic signalling. Interestingly, PKR1 and PKR2 are 85% identical and use G protein signalling pathway. The remaining question is whether these two receptors are coupled to different Gq family proteins, thereby inducing different signalling pathways.

Prokineticins/EG-VEGFs have been shown to be powerful angiogenic and chemotactic stimulators.1,4,6 Prokineticins have been reported to induce vascular leakage in endocrine organs.1 However, receptors regulating permeability by prokineticin have not been clearly identified. Previously, we have shown that cardiac PKR1 signalling increases both angiogenesis and arteriogenesis in TG mice without inducing any pathology in cardiomyocytes and inflammatory reaction.20 The vascular phenotype of mice overexpressing PKR2 in cardiomyocytes has several features that differ from those of PKR1-induced angiogenesis. Here we showed that PKR2 overexpression in cardiomyocytes results in a striking induction in the size of blood vessels without increasing number of vessels, and enhanced vascular leakiness resulted from swollen endothelial cell ultrastructures with increased caveolae-like vacuoles and fenestrae through a paracrine mechanism. Fenestrated endothelium normally occurs in the capillaries of endocrine organs, kidney glomerulus, and visceral mucosae.38 These locations are characterized by increased filtration or increased transendothelial transport. Vascular endothelium is not fenestrated; however, in pathological conditions, endothelium becomes locally hyperpermeable by the formation of small gaps. Although the cardiac

Downloaded from by guest on December 21, 2015

Figure 6 (A) Representative illustration of zona occludens-1 (ZO-1) and PECAM-1 staining on cryosectioned hearts. Arrow shows zona occludens-1 proteins are localized on the tight junctions of the endothelial cells, which were lost in the transgenic (TG) hearts. (B) Western blot analyses for zona occludens-1 and internal control GAPDH proteins on the extracts obtained from hearts. Quantitative analyses shown in histogram revealed that zona occludens-1 levels were abolished in transgenic hearts (n¼4, P , 0.01). (C ) Zona occludens-1 localization on H5V cells treated with media conditioned by H9c2 cells overexpressing pPKR2 cDNA or control vector plasmid (p). Original magnification: 63. (D) Model for PKR2-mediated autocrine and paracrine signalling in the adult heart. Excessive level of PKR2 in cardiomyocytes induces hypertrophic growth of cardiomyocytes in an autocrine fashion and may release paracrine factor that induces endothelial cell disorganization leading to vascular leakage.

36

Acknowledgements We thank Dr K. Hu (Wurzbourg) for performing some of the functional analyses.

Conflict of interest: none declared.

Funding This work was supported by funds from the Center National de la Recherche Scientifique (CNRS) and the Universite ´ Louis Pasteur, and by grants from the Fondation de France for cardiovascular research (005326), the Fondation pour la Recherche Me ´dicale (C7100000), and the Association pour la Recherche contre le Cancer (3619ARC). K.U. is supported by fellowship from Japan Society for the Promotion of Science, D.B.D. from University of Istanbul.

References 1. LeCouter J, Kowalski J, Foster J, Hass P, Zhang Z, Dillard-Telm L et al. Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature 2001;412:877–884. 2. Li M, Bullock CM, Knauer DJ, Ehlert FJ, Zhou QY. Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle. Mol Pharmacol 2001;59:692–698. 3. LeCouter J, Lin R, Tejada M, Frantz G, Peale F, Hillan KJ et al. The endocrine-gland-derived VEGF homologue Bv8 promotes angiogenesis in the testis: localization of Bv8 receptors to endothelial cells. Proc Natl Acad Sci USA 2003;100:2685–2690. 4. LeCouter J, Zlot C, Tejada M, Peale F, Ferrara N. Bv8 and endocrine gland-derived vascular endothelial growth factor stimulate hematopoiesis and hematopoietic cell mobilization. Proc Natl Acad Sci USA 2004;101:16813–16818. 5. Dorsch M, Qiu Y, Soler D, Frank N, Duong T, Goodearl A et al. PK1/ EG-VEGF induces monocyte differentiation and activation. J Leukoc Biol 2005;78:426–434.

6. Martucci C, Franchi S, Giannini E, Tian H, Melchiorri P, Negri L et al. Bv8, the amphibian homologue of the mammalian prokineticins, induces a proinflammatory phenotype of mouse macrophages. Br J Pharmacol 2006;147:225–234. 7. Melchiorri D, Bruno V, Besong G, Ngomba RT, Cuomo L, De Blasi A et al. The mammalian homologue of the novel peptide Bv8 is expressed in the central nervous system and supports neuronal survival by activating the MAP kinase/PI-3-kinase pathways. Eur J Neurosci 2001; 13:1694–1702. 8. Ng KL, Li JD, Cheng MY, Leslie FM, Lee AG, Zhou QY. Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science 2005; 308:1923–1927. 9. Matsumoto S, Yamazaki C, Masumoto KH, Nagano M, Naito M, Soga T et al. Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2. Proc Natl Acad Sci USA 2006; 103:4140–4145. 10. Negri L, Lattanzi R, Giannini E, Colucci M, Margheriti F, Melchiorri P et al. Impaired nociception and inflammatory pain sensation in mice lacking the prokineticin receptor PKR1: focus on interaction between PKR1 and the capsaicin receptor TRPV1 in pain behavior. J Neurosci 2006;26: 6716–6727. 11. Hu WP, Zhang C, Li JD, Luo ZD, Amadesi S, Bunnett N et al. Impaired pain sensation in mice lacking prokineticin 2. Mol Pain 2006;2:35. 12. Li JD, Hu WP, Boehmer L, Cheng MY, Lee AG, Jilek A et al. Attenuated circadian rhythms in mice lacking the prokineticin 2 gene. J Neurosci 2006;26:11615–11623. 13. Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J et al. Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature 2002;417:405–410. 14. Prosser HM, Bradley A, Chesham JE, Ebling FJ, Hastings MH, Maywood ES. Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei. Proc Natl Acad Sci USA 2007;104:648–653. 15. Masuda Y, Takatsu Y, Terao Y, Kumano S, Ishibashi Y, Suenaga M et al. Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors. Biochem Biophys Res Commun 2002;293:396–402. 16. Lin DC, Bullock CM, Ehlert FJ, Chen JL, Tian H, Zhou QY. Identification and molecular characterization of two closely related G protein-coupled receptors activated by prokineticins/endocrine gland vascular endothelial growth factor. J Biol Chem 2002;277:19276–19280. 17. Soga T, Matsumoto S, Oda T, Saito T, Hiyama H, Takasaki J et al. Molecular cloning and characterization of prokineticin receptors. Biochim Biophys Acta 2002;1579:173–179. 18. Negri L, Lattanzi R, Giannini E, Melchiorri P. Bv8/Prokineticin proteins and their receptors. Life Sci 2007;81:1103–1116. 19. Ngan ES, Tam PK. Prokineticin-signaling pathway. Int J Biochem Cell Biol 2008;40:1679–1684. 20. Urayama K, Guilini C, Turkeri G, Takir S, Kurose H, Messaddeq N et al. Prokineticin receptor-1 induces neovascularization and epicardialderived progenitor cell differentiation. Arterioscler Thromb Vasc Biol 2008;28:841–849. 21. Wettschureck N, Rutten H, Zywietz A, Gehring D, Wilkie TM, Chen J et al. Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes. Nat Med 2001;7:1236–1240. 22. Nebigil CG, Maroteaux L. A novel role for serotonin in heart. Trends Cardiovasc Med 2001;11:329–335. 23. Nebigil CG, Launay JM, Hickel P, Tournois C, Maroteaux L. 5-hydroxytryptamine 2B receptor regulates cell-cycle progression: cross-talk with tyrosine kinase pathways. Proc Natl Acad Sci USA 2000; 97:2591–2596. 24. Nebigil CG, Jaffre F, Messaddeq N, Hickel P, Monassier L, Launay JM et al. Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation 2003;107:3223–3229. 25. Nebigil CG, Hickel P, Messaddeq N, Vonesch JL, Douchet MP, Monassier L et al. Ablation of serotonin 5-HT(2B) receptors in mice leads to abnormal cardiac structure and function. Circulation 2001;103: 2973–2979. 26. Nebigil CG, Etienne N, Messaddeq N, Maroteaux L. Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B receptor signaling. FASEB J 2003;17:1373–1375. 27. Nebigil CG, Choi DS, Dierich A, Hickel P, Le Meur M, Messaddeq N et al. Serotonin 2B receptor is required for heart development. Proc Natl Acad Sci USA 2000;97:9508–9513.

Downloaded from by guest on December 21, 2015

PKR2 signalling-induced vascular endothelial barrier dysfunction was associated with macrophage/monocyte infiltration across the capillary vessels, no sign of necrosis or apoptosis was observed in the cardiomyocytes, indicating that no inflammation occurred in TG heart. The mechanism involved in fenestration in TG hearts could be due to tight junctional defects in cardiac endothelial cells, which resulted from reduced ZO-1 expression. It has been shown that ZO-1 binds to the adherents junction protein, b-catenin, and the gap junction protein, connexin-43, to orchestrate tight junction complexes.39 Moreover, reduced ZO-1 levels were shown in hyperpermeabilized human coronary artery endothelial cells,40 similar to our observation in PKR2-TG hearts. Treatment of H5V endothelial cells with conditioned medium of H9c2 cells overexpressing PKR2 induced impaired endothelial cell organization and ZO-1 expression, mimicking our TG model. These data further support the involvement of paracrine factors induced by cardiomyocytePKR2 signalling to regulate vascular integrity in TG mice. PKR2-induced paracrine factors in TG hearts are currently under the investigation in our laboratory. In summary, our findings show that the overexpression of PKR2 in cardiomyocyte leads to eccentric hypertrophy by an autocrine signalling and impairs the endothelial barrier function leading to vascular leakage by a paracrine signalling (Figure 6D).

K. Urayama et al.

Fenestration and cardiac PKR2 signalling 28. Ruppel KM, Willison D, Kataoka H, Wang A, Zheng YW, Cornelissen I et al. Essential role for Galpha13 in endothelial cells during embryonic development. Proc Natl Acad Sci USA 2005;102:8281–8286. 29. Urayama K, Guilini C, Messaddeq N, Hu K, Steenman M, Kurose H et al. The prokineticin receptor-1 (GPR73) promotes cardiomyocyte survival and angiogenesis. FASEB J 2007;21:2980–2993. 30. Frantz S, Brandes RP, Hu K, Rammelt K, Wolf J, Scheuermann H et al. Left ventricular remodeling after myocardial infarction in mice with targeted deletion of the NADPH oxidase subunit gp91PHOX. Basic Res Cardiol 2006; 101:127–132. 31. Sano H, Hosokawa K, Kidoya H, Takakura N. Negative regulation of VEGF-induced vascular leakage by blockade of angiotensin II type 1 receptor. Arterioscler Thromb Vasc Biol 2006;26:2673–2680. 32. Tang CM, Insel PA. GPCR expression in the heart; ‘new’ receptors in myocytes and fibroblasts. Trends Cardiovasc Med 2004;14:94–99. 33. de Gregorio C, Curto L, Recupero A, Grimaldi P, Almoto B, Venturino M et al. Echocardiographic assessment of subclinical left ventricular eccentric hypertrophy in adult-onset GHD patients by geometric remodeling: an observational case–control study. BMC Endocr Disord 2006;6:1–8.

37 34. Nicol RL, Frey N, Pearson G, Cobb M, Richardson J, Olson EN. Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J 2001;20:2757–2767. 35. Bowman JC, Steinberg SF, Jiang T, Geenen DL, Fishman GI, Buttrick PM. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J Clin Invest 1997;100:2189–2195. 36. Takeishi Y, Ping P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh RA. Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy. Circ Res 2000;86:1218–1223. 37. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R et al. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 2000;19:6341–6350. 38. Bearer EL, Orci L. Endothelial fenestral diaphragms: a quick-freeze, deep-etch study. J Cell Biol 1985;100:418–428. 39. Harhaj NS, Antonetti DA. Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol 2004;36:1206–1237. 40. Yan S, Wang X, Chai H, Wang H, Yao Q, Chen C. Secretoneurin increases monolayer permeability in human coronary artery endothelial cells. Surgery 2006;140:243–251.

Downloaded from by guest on December 21, 2015