Ethics Statement

All experimental protocols were approved by the Animal Use and Care Committee of the University of Oulu and the Provincial Government of Western Finland Department of Social Affairs and Health. The investigations conform to the Guiding Principles for Research Involving Animals.

For human studies, the study complies with the Declaration of Helsinki. The study protocol was approved by the Internal Review Committee of the University of Semmelweis, Hungary (Ethical permission number: 5012-0/2011-EKU 142/PI/11–Semmelweis, University; Hungary) and written informed consent was obtained from the subjects, except that the Institution has the ethical permission to collect tissue samples from healthy subjects (victims of accidents).

Recombinant Adenovirus Vectors

For constructions of rat WDR12, a full-length coding region of WDR12 cDNA was cloned into the SalI and HindIII sites of the pShuttle-CMV vector (Qbiogene Inc., Illkirch, Cedex-France). The sequences for the cloning primers used were as follows; WDR12 forward 5'-GCG TCG ACC TTT CTG CCA TGG CAC AAC-3' and reverse 5'-CCC AAG CTT TCA AGC CCC CAC ATG GGA AGT-3'. The pShuttle-CMV-LacZ was a commercial plasmid (Stratagene, La Jolla, CA, USA). Adenoviruses (serotype 5) were prepared by standard protocols (Qbiogene Inc., Illkirch, Cedex-France) and purified by centrifugation on iodixanol (OptiPrep, Axis-Shield PoC AS, Oslo, Norway). The adenoviral titers (infectious unit, ifu) were determined by AdEasy Viral Titer Kit (Stratagene). Infectious unit is biologically equivalent to plaque forming unit (PFU). β -galactosidase concentration was determined by luminescent β -gal Kit (Clontech Laboratories Inc., Palo Alto, CA, USA). The recombinant adenoviruses containing the coding regions of the constitutively active mitogen-activated protein kinase kinases (MKK), MKK3b and MKK6b and wild type p38α, and p38β genes driven by CMV immediate early promoter were generated as described previously [11]. In another series of experiments, we have observed that overexpression of p38α and p38β produce similar p38 and phosho-p38-mitogen–activated protein kinase (MAPK) levels in cardiomyocytes (Kaikkonen et al., unpublished observation). MKK and p38 adenoviruses were generously supplied by Dr. Veli-Matti Kähäri from the University of Turku, Finland.

Intramyocardial Gene Transfer

We and others have previously shown that local injection of adenoviral constructs into the left ventricular free wall is an efficient site-specific method of gene delivery that targets high expression of the transgene in the LV without affecting other organs or other regions of the heart [12,13]. Male Sprague-Dawley rats weighing 250–300 g were anesthetized with medetomidine hydrochloride (Domitor, 250 μg/kg i.p.) and ketamine hydrochloride (Ketamine, 50 mg/kg i.p.). Rats were connected to the respirator through a tracheotomy. A left thoracotomy and pericardial incision was performed. Adenovirus-mediated gene transfer into the LV free wall was performed as previously described [13,14]. Different doses of adenoviral constructs were first tested to increase LV WDR12 protein levels. Then, recombinant adenovirus (4 x 10⁹ ifu), in a 100 μl volume, was injected using a Hamilton precision syringe directly into the anterior wall of the LV. The syringe was inserted in one site of the LV free wall (apex to base), and then slowly the solution was injected while withdrawing the syringe. After the operation, anaesthesia was partially antagonized with atipamezole hydrochloride (Antisedan, 1.5 mg/kg i.p.) and rats were hydrated with physiological saline solution (5 ml s.c.). For postoperative analgesia, buprenorphine hydrochloride (Vetergesic, 0.05–0.2 mg/kg s.c.) was administered. The total number of animals used was 240.

Acute Myocardial Infarction

MI was produced by ligation of the left anterior descending coronary artery (LAD) as previously described [13,15]. The sham-operated rats underwent the same surgical procedure without the ligation of LAD. Recombinant adenovirus was injected into the anterior wall of the LV before the ligation of LAD. The adenoviral gene delivery to the sham-operated hearts was performed using the same technique without the ligation of LAD.

Angiotensin II-Mediated Hypertension

Angiotensin II (Ang II, 33.3 μg/kg/h) (a generous gift from Dr. Olli Vuolteenaho, University of Oulu, Finland), was administered via subcutaneously implanted osmotic minipumps for 6 hours and 2 weeks (Alzet, Scanbur BK AB, Sollentuna, Sweden), as described previously [16,17]. Minipumps were implanted subcutaneously before the gene delivery. Ang II–mediated hypertension has been used extensively in key studies in the development of antihypertensive agents [18–21]. Using this experimental model of hypertension, mean arterial pressure increases rapidly (within 3 hours) and remains significantly elevated throughout the 2-weeks period [16]. Under our experimental conditions, Ang II type 1 receptor (AT₁-R) blockade by losartan completely abolished Ang II–induced changes in the cardiac gene expression, left ventricular weight to body weight ratio and hemodynamics [17].

Echocardiographic Measurements

Transthoracic echocardiography was performed using the Acuson Ultrasound System (Sequoia 512) and a 15-MHz linear transducer (15L8) (Acuson, MountainView, CA, USA) as previously described [13,22]. Before examination, rats were sedated with ketamine (50 mg/kg i.p.) and xylazine (10 mg/kg i.p.). Using two-dimensional imaging, a short axis view of the LV at the level of the papillary muscles was obtained, and a two dimensionally guided M-mode recording through the anterior and posterior walls of the LV was obtained. LV end-systolic and end-diastolic dimensions as well as the thickness of the interventricular septum and posterior wall were measured from the M-mode tracings. LV fractional shortening (FS) and ejection fraction (LVEF) were calculated from the M-mode LV dimensions using the Eqs 1 and 2: (1) (2)

An average of three measurements of each variable was used. All echocardiographical measurements were performed blinded by persons (E.M. and Z.Sz.), who were not aware of the treatments. After echocardiography, the animals were sacrificed. Hearts were weighed and the ventricles were immersed in liquid nitrogen and stored at—70°C for later analysis.

Extraction of Cytoplasmic Protein and Western Blot Analyses

To extract the cytoplasmic protein, the left ventricular tissue was broken and reduced to a powder in liquid nitrogen. The thawed powder was homogenized in a lysis buffer (20 mmol/l Tris-HCl (pH 7.5), 10 mmol/l NaCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l β-glycerophosphate, 1 mmol/l Na₃VO₄, 2 mmol/l benzamidine, 1 mmol/l phenylmethylsulfoxide, 50 mmol/l NaF, 1 mmol/l dithiothreitol and 10 μg/ml each of leupeptin, pepstatin and aprotinin). The cytosolic fraction was separated out by centrifugation at 2000 rpm in +4°C for 1 minute. To separate the cytoplasmic protein fraction, 5 x nuclear extraction buffer (NEB) (100 mM Tris-HCl (pH 7.5), 750 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5% Triton X 100, 12 mM sodium pyrophosphate, 5 mM β-glycerophosphate, 5 mM Na₃VO₄) was added to the tissue homogenate following by centrifugation at 12500 rpm in +4°C for 20 minutes. The supernatant was frozen in liquid nitrogen and stored in—70°C until assayed.

To extract the nuclear protein fraction, the supernatant from the first centrifugation was incubated on ice for 15 minutes, NP-40 was added, and the nuclei were collected by centrifugation at 12500 rpm for 30 seconds. The pellet was resuspended in a solution containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1mM EGTA, 1 mM Na₃VO₄, 2 mM benzamidine, 1 mM PMSF, 50 mM NaF, 1 mM DTT, 3 μg/ml 1-chloro-3-tosylamido-7-phenyl-2-butanone (TPCK), 3 μg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone (TLCK), and 10 μg/ml each of leupeptin, pepstatin and aprotinin. The samples were incubated at +4°C for 30 minutes, centrifuged at 12500 rpm for 5 minutes and the resulting supernatants frozen in liquid nitrogen and stored in—70°C until assayed. All protein concentrations were determined by Bio-Rad protein assay.

In the western blot analysis, 30 μg total protein or nuclear protein was subjected to SDS-PAGE and the separated proteins were electrically transferred to Optitran BA-S 85 nitrocellulose membranes (Scleicher & Schuell BioScience, Dassel, Germany). After blocking the nonspecific background in a mixture (1:1) of Odyssey Blocking Buffer—Tris–buffered saline (TBS), nitrocellulose membranes were incubated with appropriate primary antibody in Odyssey Blocking Buffer–TBS overnight at +4°C. Nitrocellulose membranes were incubated with antiphospho-p38-MAPK, antiphospho-ERK1/2, antisarcoplasmic reticulum Ca²⁺-ATPase (SERCA2), anti-p38-MAPK or anti-ERK1/2, anti-WDR12, anti-BOP1, anti-Pescadillo, anti-phospho-heat shock protein 27 (HSP27), anti-HSP27, anti-Lamin B and anti-GAPDH-antibody. The antibody dilution varied from 1:200 to 1:2000, depending on the signal strength. After washing, antibody binding was detected with specific secondary antibodies, fluorescent (Alexa Fluor) antibodies (1:3000–1:5000) used with Odyssey Infrared Imaging System.

For a second western blot, the membranes were stripped in buffer containing 62.5 mmol/l tris (pH 6.8), 2% sodium dodecyl sulfate, and 100 mmol/l mercapthoethanol. The detection was based on fluorescence measured by the Odyssey Infrared Imaging System. The bands were scanned and analyzed with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Antibodies were obtained from Millipore (Temecula, CA, USA), Invitrogen (Carlsbad, CA, USA), Aviva systems biology (San Diego, CA, USA), Santa Cruz Biotechnology (CA, USA) and Cell Signaling Technology (Beverly, MA, USA).

Isolation and Analysis of RNA

The RNA was extracted from the left ventricular tissue by using the guanidine-thiocyanate-CsCl method [23]. Cyclin-dependent kinase 4 inhibitor 2c (Cdkn2c), p19ARFexon1b, p16INK4a, cyclin-dependent kinase 4 inhibitor 2b (Cdkn2b), tropomyosin 1, WDR12, fibronectin-1 and 18S mRNA levels were analyzed by the RT-PCR using TaqMan chemistry on an ABI 7300 Sequence Detection System (Applied Biosystems) as previously described [24–26]. The sequences of the forward and reverse primers and for fluorogenic probes for RNA detection are shown in Table 1. The results were normalized to 18S RNA quantified from the same samples.

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Table 1. Forward and reverse primer and fluorogenic probe sequences used for real time quantitative RT-PCR analysis.

https://doi.org/10.1371/journal.pone.0124907.t001

Histology, Immunohistochemistry and Image Analysis

For histological analysis, the LVs were fixed in 10% buffered formalin solution. Transversal sections of the LV were embedded in paraffin, and 5-μm sections were cut. Sections were cut from the mid-section of the heart, at the level of the papillary muscles. Samples from different animals were obtained in an identical way and from the corresponding sites in order to make the samples fully comparable. Sections were stained with hematoxylin and eosin or Massons's trichrome to examine the fibrotic area of the LV. Previously we have studied the local response to adenovirus-mediated gene transfer by measuring the fibrotic area in the LV at 2 weeks after intramyocardial injection of adenoviral construct expressing LacZ and PBS-based buffer (3%-iodixanol-PBS) as well as from the hearts with needle-stick (no injection of fluid) and non-injected hearts. The results demonstrated that the degree of fibrosis did not differ between PBS-based buffer- and LacZ-injected hearts, but tended to be higher in these groups than in non-injected hearts [27].

Pecam-1 (sc-1506-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to stain endothelial cells. The number of capillaries was calculated from five representative high power fields (40×) from the LV of each section; 3 from epicardial and 2 from endocardial side of the LV were selected. To detect apoptotic cells, in situ labeling of the 3'-ends of the DNA fragments generated by apoptosis-associated endonucleases was performed using the ApopTag in situ apoptosis detection kit (Chemicon, Temicula, CA, USA), as previously described [28]. Briefly, DNA fragmentation was identified by applying terminal transferase enzyme with digoxigenin-labeled nucleotides. Anti-digoxigenin antibody was used to recognize the digoxigenin-labeled nucleotide chains attached to the 3'-ends of sample DNA. A color reaction was produced with diaminobenzidine and the sections were counterstained with hematoxylin. The apoptotic cells and bodies were counted in 5 high power fields (40× objective) choosing hot spot areas in each sample in order to make the results comparable. To determine whether TUNEL positive cells and proliferating cells were cardiomyocytes, cardiac fibroblasts or endothelial cells, immunofluorescence stainings were performed with alpha-actinin (ab28052, Abcam Inc., Cambridge, MA, USA), anti-prolyl 4-hydroxylase β (MAB2073, Millipore, Temecula,CA, USA) or Pecam-1 (sc-1506-R, Santa Cruz Biotechnology), respectively, and DAPI (diamidinophenylindole dihydrochloride, Sigma-Aldrich, St. Louis, MO, USA) as a nuclear stain.

To examine the efficiency and localization of the WDR12 gene delivery, the sections were incubated with specific polyclonal anti-WDR12 antibody (sc-132875, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at the dilution of 1:50 3 days after gene transfer. Primary antibody for c-kit (sc-168, Santa Cruz Biotechnology, CA, USA) was used to stain stem cells. The number of c-kit+ cells in the anterior wall of LV was counted. The area of counted section was examined by computerized methods and a number of positively staining cells was related to the area (cells/35 mm²). To identify cells undergoing division, immunohistochemical labeling of nuclear Ki-67 or Phospho-histone-H3 antigen was performed by using monoclonal mouse anti-rat Ki-67 antigen antibody (DakoCytomation, Glostrup, Denmark) or Phospho-histone-H3 antigen antibody (Cell Signaling Technology, Beverly, MA, USA). The whole LV was scanned and stained cells were counted from high power fields (40×) choosing 5 hot spot areas in each sample. The primary antibodies were detected by peroxidase conjugated EnVision Detection Kit system (DakoCytomation, Denmark) and the samples were counterstained with haematoxylin. All measurements were performed blinded by persons, who were not aware of the treatments.

Cell Culture

Neonatal rat ventricular myocytes were prepared from 2- to 4-day-old Spraque-Dawley rats as described earlier [29]. WDR12 or LacZ-adenoviruses were added to the cell culture medium 24h after the cells were plated and incubated 24h, at the virus concentration 6 MOI. The media were replaced every 24h, and 48h after infection, the cells were washed twice with PBS and quickly frozen at ‒70°C. For mechanical stretch experiments cardiomyocytes were plated on Bioflex plates and exposed to cyclic mechanical stretch as previously described [30]. Mechanical stretch was applied by Flexercell strain unit FX-5000. Adenoviral gene transfer of p38α and p38β as well as WDR12 in neonatal cardiomyocytes was performed as described previously [31].

Cardiac fibroblasts were separated from myocytes and plated on 10-cm dishes. On reaching confluences, fibroblasts were passaged into 6- or 12-well plates (170,000 or 70,000 cells/well, respectively). Specific siRNAs (WDR12 siRNA1 5’-GGGCCAGUUUCUUCGAAUGtt-3’ and WDR12 siRNA2 5’-GGAAAUAAGGUGUUCAAUUtt-3’) from Ambion, and negative control siRNA (mixed Ambion and Sigma-Aldrich) were transfected into the cardiomyocytes or cardiac fibroblasts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as transfection reagent. Cells were incubated in OPTI-mem (Invitrogen, Carlsbad, CA, USA) for 24h and thereafter cells were incubated in CSFM (myocytes) or DMEM+10% FBS (fibroblasts).

Total RNA was isolated from cell culture neonatal rat ventricular myocytes with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol by using the Phase Lock Gel system (Eppendorf AG, Hamburg, Germany). Cultured cardiomyocytes were lysed in ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄) supplemented with protease-inhibitor cocktail, phosphatase-inhibitor cocktail and 1mM DTT. The lysate was vortexed and then cleared by centrifugation at +4°C. The supernatant was then transferred in a new tube as the total protein extract.

The nuclear and cytosolic proteins were extracted adapting the protocol described by Schreiber et al. [32]. Cultured cardiomyocytes were washed and scraped with ice-cold 1x phosphate-buffered saline. After centrifugation the cell pellets were resuspended in 100 μl of low salt buffer consisting of 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA and 0.1 mM EGTA supplemented with protease-inhibitor cocktail, phosphatase-inhibitor cocktail and 1 mM DTT. The suspensions were then allowed to swell on ice for 15 min. Then, membrane proteins were solubilised and isolated by adding 10 μl of 10% IGEPAL CA-630 detergent and vortexing vigorously followed by centrifugation. The supernatants were collected as the cytosolic fragments. The pellets were resuspended of high salt buffer containing 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA and 1 mM EGTA, with supplements similar to those in low salt buffer and then rocked for 15 min. The samples were centrifuged and the supernatant was collected as the nuclear fragment. The entire procedure was carried out +4°C. Protein concentrations were determined with the Bio-Rad protein assay.

Human LV Tissue Samples

LV samples were obtained from organ donors (n = 2) and patients with end-stage heart failure due to cardiomyopathy (n = 4) (2 patients were diagnosed with dilated cardiomyopathy, 1 with hypertrophic cardiomyopathy and 1 with idiopathic cardiomyopathy). The donor subjects had no cardiac history and no significant structural abnormalities on cardiac donor screening using echocardiography and coronary angiography. Donors were maintained under intensive care, but hearts were excluded ultimately from heart implantation because of age, size mismatch or blood type incompatibilities. After removal, cardiac tissue samples were blotted dry, immersed in liquid nitrogen, and stored at ‒80°C until assayed.

All patients were classified according to New York Heart Association (NYHA) functional classes III and IV. Transthoracic echocardiograms were performed using a 2.5-MHz linear transducer. Coronary angiographies were performed in all subjects using percutaneous femoral or radial approach by the Judkins technique. General patient characteristics are shown in Table 2.

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Table 2. LV tissue samples were obtained from explanted human hearts in patients with cardiomyopathy undergoing heart transplantation.

https://doi.org/10.1371/journal.pone.0124907.t002

Relationship between MI Associated WDR12 Allele and Cardiac Function

Malmö Preventive Project (MPP) started in the mid 1970s at the Malmö University Hospital, Malmö, Sweden as a prospective population-based study [33]. Between 1974 and 1992 a total of 33,346 men and women from the Malmö city area were recruited and screened for conventional risk factors for all-cause mortality and cardiovascular disease (CVD). Briefly, specially trained nurses performed the examinations in the morning. Participants were fasting overnight prior to investigation. Height and weight were measured in light indoor clothing without shoes. Blood samples were collected and biochemical parameters were analyzed by routine methods at the Department of Clinical Chemistry, Malmö University Hospital. Afterwards, participants answered a questionnaire consisting of approximately 260 questions using a self-administered computer program. The questions centered on personal and family history of CVD (myocardial infarction and stroke), hypertension, diabetes and cancer, as well as on conventional risk factors for these diseases. Antihypertensive treatment was defined as a positive answer to the following question: Do you take medication for high blood pressure? Blood pressure (BP) was measured in the supine position by specially trained nurses with a mercury sphygmomanometer and an appropriate cuff placed around the right arm at the level of the heart. In the years 2002–2006, all survivors from the original MPP cohort were invited to a follow-up examination. Of these, 18,240 participants responded to the invitation and were re-examined [34]. There were no exclusion criteria and of those who were alive the participation rate was 72%. For genetic studies, additional samples of peripheral venous blood were collected, as the original MPP design did not encompass DNA analyses.

In a sub-sample of the MPP follow-up examination, 1792 participants underwent echocardiography on a separate visit. As described in the original publication [35], subjects undergoing echocardiography were randomly selected from groups of glucometabolic status (normal fasting glucose, impaired fasting glucose, new onset diabetes mellitus and prevalent diabetes mellitus), with oversampling of the groups with glucometabolic disturbances). All subjects were studied in the left lateral decubital position. Conventional echocardiography/Doppler and tissue Doppler imaging (TDI) were conducted with a 3V2c transducer (Acuson Sequoia, Mountain View, CA, USA) or a S3 transducer (Sonos 5500 Philips, Andover, MA, USA). Parasternal long- and short-axis, and apical four- and two-chamber views were used to evaluate cardiac dimensions and LVEF [36]. Diastolic heart function was defined according to European Society of Cardiology guidelines [37] and we compared genotypic distribution of the previously myocardial infarction associated WDR12 SNP rs6725887 [5] between subjects who had normal diastolic function or only mild diastolic dysfunction (relaxation impairment) and those with significant diastolic dysfunction (i.e. subjects displaying a pseudonormalization pattern or a restrictive pattern). Left atrial area, indexed for body surface area, was used as a continuous trait for diastolic function. All analyses were performed offline without any knowledge of the subjects’ clinical status. Out of the 1792 subjects who underwent echocardiography, diastolic function could be determined in 1636 subjects and left atrial size in 1681 subjects. DNA was available and extracted from buffy coats with the use of QIAamp-96 spin blood kits (QIAGEN, Stockholm, Sweden) from 1496/1636 and 1531/1681 subjects and the rs6725887 SNP was successfully genotyped in 1394/1496 and 1436/1531 subjects using primers and probes which were custom synthesized by Applied Biosystems (Foster City, CA, USA) according to standard recommendations for the AB Prism 7900HT analysis system, and genotyped with polymerase chain reaction-based TaqMan method [38]. Detailed clinical characteristics of the 1436 subjects included in the genetic analyses of left atrial area are shown in Table 3.

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Table 3. Clinical characteristics of the subjects included in the genetic analyses of left atrial area in Malmö Preventive Project (MPP).

https://doi.org/10.1371/journal.pone.0124907.t003

Statistics

Results are expressed as mean±SEM. For statistical analyses, the data were first tested by using the Shapiro-Wilk test for normality. Because all variables were normally distributed, statistical significance was evaluated by 1-way ANOVA followed by a least significant difference (LSD) post hoc test for multiple comparisons. The Student t test was used for comparison between 2 groups. A probability value of <0.05 was considered statistically significant.

Crude and age- and sex-adjusted logistic regression models were used to relate rs6725887genotypes to a dichotomous dependent variable of diastolic dysfunction (presence of pseudonormalization pattern or restrictive pattern versus normal diastolic function or relaxation impairment). In crude and age- and sex-adjusted linear regression models we related rs6725887 genotypes to body surface normalized left atrial size and ejection fraction as a continuous variable.

Cardiac Overload Induces WDR12 Expression through Direct Myocyte Stretch and p38 MAPK

Because WDR12 was associated with early-onset MI in GWAS studies [4,5], we first assessed the effect of post-infarction remodeling process on LV WDR12 gene expression. In response to MI induced by ligation of LAD in rats, WDR12 mRNA levels increased 2.2-fold (Fig 1A) and WDR12 protein levels 1.6-fold (Fig 1B) at day 1. When the effect of pressure overload on LV WDR12 expression was studied by infusing Ang II up to 2 weeks in rats, WDR12 mRNA levels were significantly elevated at 6 hours (1.9-fold)(Fig 1C). To characterize the potential mechanisms by which cardiac overload increases WDR12 expression, we employed an in vitro mechanical stretch model of cultured cardiomyocytes [39]. As shown in Fig 1D, direct mechanical stretch augmented WDR12 gene expression in neonatal rat cardiomyocytes, peaking at 12 h (2.6-fold). Moreover, once the role of the stretch-activated p38 MAPKs [25] in regulating WDR12 expression was studied, p38α but not p38β gene transfer in vitro increased WDR12 mRNA levels 1.8-fold (Fig 1E). To examine the LV localization of WDR12 we used immunofluorescence analysis with rat thymus [8] as a positive control. WDR12 was expressed in cardiac myocytes of rat hearts at 1 day post-infarction (Fig 1F and 1G).

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Fig 1. Induction of WDR12 expression by cardiac overload.

A. Up-regulation of LV WDR12 mRNA, and B. protein levels by experimental MI at day 1 in rats. Representative Western blots are shown. Bands were detected from the same gel. Results are expressed as mean±SEM (n = 11 to 16). **P<0.01 MI (solid bars) versus sham (open bars) group (Student t test). C. Up-regulation of WDR12 mRNA levels in Ang II-induced hypertension at 6 hours. Results are expressed as mean±SEM (n = 6 to 8). **P<0.01 Ang II (solid bars) versus control (open bars) group (Student t test). D. Increase of WDR12 mRNA levels by mechanical stretch (n = 5 to 6), and E. p38 MAPK gene transfer (n = 6) in vitro in cell culture. The results are expressed as mean±SEM. *P<0.05, **P<0.01 versus control-group (1-way ANOVA followed by LSD post hoc test). F. Immunofluorescence staining of LV anterior wall, and G. thymus at day 1 post-infarction in rats. WDR12 is located in cardiac myocytes. WDR12 (green) and DAPI (blue).

https://doi.org/10.1371/journal.pone.0124907.g001

Augmentation of LV WDR12 Levels by Adenoviral Gene Delivery

To study the direct myocardial effects of WDR12, we established an in vivo gene transfer protocol to locally increase LV WDR12 levels in the adult rat heart. As shown in Fig 2A, a dose-dependent increase in WDR12 protein levels, measured by western blot, was noted at day 3 after gene delivery. When WDR12 expressing adenoviral construct was injected into the LV free wall at 4x10⁹ ifu, WDR12 mRNA levels (Fig 2B) were highest at 1 week and protein levels (Fig 2C) at day 3 after injections and decreased thereafter to the levels of LacZ-treated hearts at 2 weeks. The increase in LV WDR12 protein levels at 1 week after gene delivery closely mimicked those increases observed in rats post-infarction and in response to pressure overload (see Fig 1B and 1C). The efficiency and localization of the WDR12 gene delivery was further confirmed by immunofluorescence. As shown in Fig 2D, immunofluorescence analysis showed augmented local staining in the cardiomyocyte nuclei of WDR12-treated hearts compared to LacZ-treated hearts.

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Fig 2. Cardiac specific activation of WDR12 by adenoviral gene delivery into the LV.

A. Different doses of WDR12 adenoviral constructs were tested to increase LV WDR12 protein levels. Representative Western blots are shown. Bands were detected from the same gel. The results are expressed as mean±SEM (n = 3). *P<0.05, **P<0.01 versus 1x10⁹ IFU/rat injected group (1-way ANOVA followed by LSD post hoc test). B. WDR12 mRNA levels measured by RT-PCR, and C. WDR12 protein levels assessed by Western Blot analyses from the tissue samples 3 days, 1 week and 2 weeks after WDR12 gene delivery. Bands were detected from the same gel. Open bars represent LacZ and solid bars WDR12. The results are expressed as mean±SEM (n = 8 to 10). *P<0.05, **P<0.01, ***P<0.001 versus LacZ (Student t test). D. Immunofluorescence staining of LV anterior wall at day 3 after gene transfer showing WDR12 predominantly in cardiomyocytes (WDR12, red).

https://doi.org/10.1371/journal.pone.0124907.g002

Local Myocardial WDR12 Gene Delivery Deteriorates Cardiac Function in Normal Adult Rats

The effect of WDR12 gene delivery on cardiac function was analyzed by echocardiography (Fig 3A through 3G and Table 4) at 1 week following gene transfer. WDR12 gene transfer in normal rat hearts resulted in reduced interventricular septum thickness (Fig 3B and 3C) and increased LV diameters (Fig 3D and 3E) in both diastole and systole. Moreover, WDR12 gene delivery significantly decreased LVEF (P<0.05) and FS (P<0.05) (Fig 3F and 3G), collectively indicating that WDR12 gene delivery deteriorated cardiac function in normal adult rats.

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Fig 3. Intramyocardial WDR12 gene delivery deteriorates cardiac function in normal adult rat.

Adenoviral construct expressing WDR12 and LacZ were injected into LV free wall. Echocardiographic measurements were performed 1 week after gene transfer. A. Representative M-mode images are shown. Treatment with WDR12 overexpressing vector significantly decreased interventricular septum diastolic (B) and systolic thickness (C), and increased LV diastolic (D) and systolic diameters (E). Overexpression of WDR12 decreased LV ejection fraction (F) and fractional shortening (G). The results are expressed as mean±SEM (n = 7 to 10). *P<0.05, **P<0.01 versus LacZ-control group (Student t test).

https://doi.org/10.1371/journal.pone.0124907.g003

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Table 4. Effect of intramyocardial WDR12 gene delivery on cardiac structure and function in normal adult rat hearts, post-infarction, and in angiotensin II-induced hypertension at 1 week.

https://doi.org/10.1371/journal.pone.0124907.t004

Effects of WDR12 Gene Delivery Post-Infarction and in Angiotensin II-Induced Hypertension

Because both MI and pressure overload caused significant up-regulation of WDR12 levels, we next examined the effects of WDR12 gene transfer on LV function and structure in MI induced by ligation of LAD and in angiotensin II-induced hypertension in rats. LV WDR12 mRNA and protein levels at 1 and 2 weeks after WDR12 gene transfer post-infarction and in Ang II-mediated hypertension increased similarly to those observed in normal adult rat hearts (Data not shown). As assessed by echocardiography, LV dimensions, LVEF and FS of WDR12-treated animals were similar to those of LacZ-treated hearts, while ligation of LAD alone caused a marked decrease in LVEF and FS (Table 4). Likewise, WDR12 gene transfer did not significantly affect LV dimensions, LVEF or FS in rats infused with Ang II (Table 4). However, evaluation of diastolic function revealed that WDR12 gene transfer decreased E/A ratio by 32% (P<0.05) at 1 week post-infarction (Fig 4A and 4B), suggesting that WDR12 gene delivery results in diastolic dysfunction. There was also a non-significant decrease in E/A ratio in normal rat hearts (13%) and in Ang II-mediated hypertension (27%)(Fig 4B). There was also a tendency towards increased isovolumic relaxation time (IVRT) at 1 week (Fig 4C). Interestingly, consistently with the impairment of cardiac function, LV SERCA2 protein levels were lower after WDR12 gene transfer at day 3 in normal adult rats, but remained unchanged in WDR12-treated hearts post-infarction and in Ang II-mediated hypertension (Fig 5A through 5C). Furthermore, SERCA2 mRNA levels decreased in response to adenoviral transfection of WDR12 in vitro in rat neonatal cardiomyocytes and increased by cell-specific silencing of WDR12 (Fig 5D and 5E).

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Fig 4. Intramyocardial WDR12 gene delivery decreases diastolic function in rats.

Adenoviral construct expressing WDR12 and LacZ were injected into LV free wall. Echocardiographic measurements were performed 1 week after WDR12 gene transfer. A. Representative mitral flow velocity images of LacZ- and WDR12-injected infarcted hearts are shown. B. E/A ratio decreased significantly by intramyocardial WDR12 gene transfer in infarcted hearts, and non-significantly in WDR12-treated normal hearts and Ang II-infused hearts. C. IVRT values increased non-significantly by intramyocardial WDR12 gene transfer, in WDR12-treated normal hearts, infarcted hearts and Ang II-infused hearts. The results are expressed as mean±SEM (n = 7 to 10). *P<0.05 versus LacZ-control group (Student t test).

https://doi.org/10.1371/journal.pone.0124907.g004

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Fig 5. SERCA2 expression after intramyocardial WDR12 gene transfer and after WDR12 gene silencing.

SERCA2 protein levels were assessed by Western Blot analyses from the LV samples 3 days, 1 week and 2 weeks after WDR12 gene delivery in normal adult rats (A), 1 week after myocardial infarction (B) and in angiotensin II (Ang II)-induced hypertension at 1 week (C) (n = 8 to 10). Bands were detected from the same gel. D. SERCA2 mRNA levels after adenoviral transfection of WDR12 in vitro in rat neonatal cardiomyocytes (n = 4). A. open bars represent LacZ and solid bars WDR12. *P<0.05, **P<0.01 versus LacZ (Student t test). E. SERCA2 mRNA levels after WDR12 siRNA transfection in rat neonatal cardiomyocytes (n = 4). *P<0.05, **P<0.001 versus negative control (1-way ANOVA followed by LSD post hoc test). The results are expressed as mean±SEM.

https://doi.org/10.1371/journal.pone.0124907.g005

Marked Reduction of LV Cell Proliferation by WDR12 Overexpression

Since PeBoW complex regulates cell proliferation [9], we next assessed whether the changes in cardiac structure and function after WDR12 gene delivery were related to the alterations in cell proliferation by staining histological sections immuno-histochemically against nuclear Ki-67. As shown in Fig 6A, intramyocardial WDR12 gene delivery resulted in a marked decrease (75%) in the number of Ki-67 positive cells at 1 week. Double immunofluorescence staining of proliferating cells showed that they were positive for fibroblast marker prolyl 4-hydroxylase (Fig 6A) but not for cardiomyocyte or endothelial cell markers (Fig 7A and 7B). We also analyzed histological sections for apoptotic cell death, myocardial fibrosis, cardiac stem cell recruitment and angiogenesis. As assessed by TUNEL assay, WDR12 gene transfer significantly decreased apoptotic cell death (65%)(Fig 6B). Double immunofluorescence staining of TUNEL+ cells showed that also they were positive for fibroblast marker prolyl 4-hydroxylase (Fig 6B) but not for cardiomyocyte or endothelial cell markers (Fig 7C and 7D). Moreover, there was a non-significant increase in myocardial fibrosis, measured by staining histological sections with Masson’s trichrome (Fig 6C), and a significant increase in LV expression of a fibrosis-related gene, fibronectin-1 (Fig 6D). On the other hand, the number of c-kit+ cells or coronary angiogenesis did not significantly differ between LacZ- and WDR12-treated hearts (Data not shown). Finally, because dominant-negative mutant of WDR12 has been shown to block rRNA processing, induce a reversible cell cycle arrest, and trigger accumulation of p53 in a p19ARF-independent manner in proliferating cells [9], we examined the effects of WDR12 gene delivery on the LV mRNA levels of selected regulators of the cell cycle. However, no significant differences in cyclin-dependent kinase inhibitor 2b and 2c, p19ARF and p16INK4a mRNA levels were observed between WDR12-treated and LacZ-treated normal rat hearts and post-infarction at 1 week after gene transfer (Fig 8).

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Fig 6. Myocardial cellular proliferation, apoptotic cell death and fibrosis in the LV after local WDR12 gene delivery at 1 week.

A. Immunohistochemical staining against Ki-67 was performed to study the effect of WDR12 gene transfer on cellular proliferation. The whole left ventricle was scanned and stained cells were counted from high power fields (40x) choosing 5 hot spot areas in each sample. Immunofluorescence staining showing that proliferating cells were cardiac fibroblasts (red, Phospho-histone-H3; green, Prolyl 4-hydroxylase β). B. The rate of apoptosis was assessed by TUNEL. Immunofluorescence staining showing that apoptotic cells were cardiac fibroblasts (Red, TUNEL; green, Prolyl 4-hydroxylase β). C. Masson’s trichrome staining was used to define area of fibrosis by computerized methods. The results are expressed as mean±SEM (n = 4 to 5). D. Fibronectin-1 mRNA levels were increased 1 week after WDR12 gene delivery. Open bars represent LacZ and solid bars WDR12. The results are expressed as mean±SEM (n = 8 to 10). *P<0.05, **P<0.01 versus LacZ (Student t test).

https://doi.org/10.1371/journal.pone.0124907.g006

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Fig 7. Immunofluorescence staining showing that proliferating cells and apoptotic cells were non-cardiomyocytes and non-endothelial cells.

A. Proliferating cells (red, Phospho-histone-H3; green, α-actinin). B. Proliferating cells (red, Phospho-histone-H3; green, Pecam-1). C. Apoptotic cells (red, TUNEL, green, α-actinin), D. Apoptotic cells (red, TUNEL, green, Pecam-1).

https://doi.org/10.1371/journal.pone.0124907.g007

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Fig 8. Effects of WDR12 gene delivery on the expression of putative WDR12 target genes in normal rat heart and during 1 week post-infarction.

A. Cyclin-dependent kinase inhibitor 2 b (Cdkn2b), B. Cdkn2c, C. p19ARFexon1b, D. p16INK4a mRNA levels in normal rat heart and E. Cdkn2b, F. Cdkn2c, G. p19ARFexon1b, H. p16INK4a mRNA levels at 1 week post infarction. A-D. Open bars represent LacZ and solid bars WDR12. The results are expressed as mean±SEM (n = 7–10). P = ns versus LacZ (Student t test).

https://doi.org/10.1371/journal.pone.0124907.g008

Regulation of BOP1 and Pes Protein Levels by WDR12

Because WDR12 forms a stable complex and directly interacts with nucleolar target genes Pes1 and BOP1 in rRNA processing and proliferation [40,41], we next examined protein levels of BOP1 and Pes after WDR12 gene transfer by Western blot analysis. Remarkably, WDR12 overexpression consistently increased BOP1 in normal adult rat hearts (P<0.05)(Fig 9A), post-infarction (P<0.05)(Fig 9C), in Ang II-induced hypertension (P<0.05)(Fig 9E) and in vitro in neonatal rat cardiomyocytes (p = 0.061)(Fig 9G and Fig 10A and 10B) and in cardiac fibroblasts (Fig 11A and Fig 12A and 12B). Pes protein levels increased significantly by WDR12 gene transfer only at 1 week post-infarction (Fig 9B, 9D, 9F and 9H and Fig 11B). Moreover, BOP1 and Pes protein levels were decreased after WDR12 siRNA transfection in both neonatal cardiomyocytes (Fig 9I and 9J and Fig 10C and 10D) and fibroblasts (Fig 11C and 11D and Fig 12C and 12D).

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Fig 9. Effects of intramyocardial WDR12 gene delivery and WDR12 gene silencing on BOP1 and Pes nuclear protein expressions.

A. BOP1, and B. Pes protein expression increased in normal rat hearts 1 week after gene transfer. C. BOP1, and D. Pes proteins were up-regulated by WDR12 gene transfer in infarcted hearts. E. BOP1 protein expression increased in Ang II-induced hypertension, but no differences in Pes protein level was noted (F). G. and H. BOP1 and Pes protein levels after adenoviral transfection of WDR12 in vitro in rat neonatal cardiomyocytes. The results are mean±SEM (n = 7 to 10). *P<0.05 versus LacZ-control group (Student t test). I. and J. BOP1 and Pes protein levels after WDR12 siRNA transfection in rat neonatal cardiomyocytes (n = 4). *P<0.05, **P<0.01 versus negative control (1-way ANOVA followed by LSD post hoc test). Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g009

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Fig 10. Activation and silencing of WDR12 in rat neonatal cardiac myocytes in vitro.

A. WDR12 mRNA levels measured by RT-PCR, and B. WDR12 protein levels assessed by Western Blot analyses from total protein cell lysates 48 hours after WDR12 gene transfer (n = 8). ***P<0.001 versus LacZ-group (Student t test). C. WDR12 mRNA and D. WDR12 protein levels after WDR12 siRNA transfection (n = 4). **P<0.01, ***P<0.001 versus negative control group (1-way ANOVA followed by LSD post hoc test). The results are expressed as mean±SEM. Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g010

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Fig 11. Effects of WDR12 gene delivery and WDR12 gene silencing in vitro in rat neonatal fibroblasts.

A. BOP1 and B. Pes protein levels after adenoviral transfection of WDR12. *P<0.05 versus LacZ-control group. C. BOP1 and D. Pes protein levels after WDR12 siRNA transfection *P<0.05, **P<0.01 versus negative control group (1-way ANOVA followed by LSD post hoc test). The results are mean±SEM (n = 4). Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g011

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Fig 12. Activation and silencing of WDR12 in rat neonatal cardiac fibroblasts in vitro.

A. WDR12 mRNA levels measured by RT-PCR, and B. WDR12 protein levels assessed by Western Blot analyses from total protein cell lysates after WDR12 gene transfer. ***P<0.001 versus LacZ-group (Student t test). C. WDR12 mRNA and D. WDR12 protein levels after WDR12 siRNA transfection (n = 4). *P<0.05, **P<0.01, ***P<0.001 versus negative control group (1-way ANOVA followed by LSD post hoc test). The results are expressed as mean±SEM (n = 4). Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g012

WDR12 activates p38 MAPK/HSP27 and ERK1/2 Pathways

To further evaluate potential mechanisms triggering structural changes and LV dysfunction by local WDR12 gene delivery, we assessed changes in the phosphorylations of p38 MAPK, ERK1/2 and HSP27, involved in regulation of cell proliferation, differentiation and survival [42,43]. WDR12 gene delivery significantly increased both p38 MAPK/HSP27 and ERK1/2 phosphorylations in the adult rat hearts at 1 week (Fig 13A through 13C). Up-regulation of WDR12 levels produced increase of p38 MAPK and HSP27 but not ERK1/2 phosphorylation also post-infarction (Fig 13D through 13F). In Ang II-induced hypertension, WDR12 gene delivery significantly increased both p38 MAPK and ERK1/2 phosphorylations and non-significantly HSP27 phosphorylation (Fig 13G through 13I). Finally, p38 MAPK, HSP27 and ERK1/2 phosphorylations were up-regulated after adenoviral transfection of WDR12 in vitro in rat neonatal cardiomyocytes (Fig 13J through 13L) and fibroblasts (Fig 14A, 14C and 14E) associated with a 17.8-fold increase in WDR12 mRNA and 8.6-fold increase in WDR12 protein levels in myocytes (Fig 10A and 10B) and 4.5-fold increase in WDR12 mRNA and 5.2-fold increase in WDR12 protein levels in fibroblasts (Fig 12A and 12B). Furthermore, WDR12 siRNA gene silencing in vitro resulted in decreased phosphorylation of p38 MAPK, HSP27 and ERK1/2 in neonatal myocytes (Fig 13M, 13N and 13O and Fig 10C and 10D) and in fibroblasts (Fig 14B, 14D and 14F and Fig 12C and 12D).

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Fig 13. Regulation of p38 MAPK, HSP27 and ERK1/2 phosphorylations by WDR12.

A. and B. p38 MAPK and HSP27 phosphorylations were up-regulated in normal heart, D. and E. after infarction, and G. and H. p38 MAPK in Ang II-induced hypertension at 1 week after WDR12 gene delivery. C. ERK1/2 phosphorylations were up-regulated in normal heart at 3 days, and I. in Ang II-induced hypertension at 1 week, but not post-infarction (F). J. K. and L. p38 MAPK, HSP27 and ERK1/2 phosphorylations were increased by adenoviral transfection of WDR12 in vitro in rat neonatal cardiomyocytes (n = 7 to 10). A-C. open bars represent LacZ and solid bars WDR12. *P<0.05, **P<0.01, ***P<0.001 versus LacZ-control group (Student t test). M. N. and O. p38 MAPK, HSP27 and ERK1/2 phosphorylations were decreased by WDR12 siRNA transfection in vitro in rat neonatal cardiomyocytes (n = 4). *P<0.05 versus negative control group (1-way ANOVA followed by LSD post hoc test). The results are mean±SEM. Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g013

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Fig 14. Regulation of p38 MAPK, HSP27 and ERK1/2 phosphorylation by WDR12 in vitro in rat neonatal cardiac fibroblasts.

A. C. and E. p38 MAPK, HSP27 and ERK1/2 phosphorylations were increased by adenoviral transfection of WDR12. *P<0.05, ***P<0.001 versus LacZ-control group (Student t test). B. D. and F. p38 MAPK, HSP27 and ERK1/2 phosphorylations were decreased by WDR12 siRNA transfection in vitro in rat neonatal cardiac fibroblasts. *P<0.05, **P<0.01 versus negative control group (1-way ANOVA followed by LSD post hoc test). The results are mean±SEM (n = 4). Representative Western blots are shown. Bands were detected from the same gel.

https://doi.org/10.1371/journal.pone.0124907.g014

Augmented LV WDR12 Expression in Patients with cardiomyopathy

WDR12 protein was also found to be expressed in the LV of control human hearts and in patients with cardiomyopathy (Fig 15A and 15B). Nuclear WDR12 protein levels were higher (1.8-fold) in patients with cardiomyopathy than in control hearts (Fig 15B and 15C), and there was also a non-significant increase (2.1-fold) in total WDR12 protein levels in patients with cardiomyopathy when compared to control hearts (Fig 15A and 15C). Also WDR12 mRNA as well as ANP and BNP mRNA levels were higher in patients with cardiomyopathy (Data not shown).

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Fig 15. WDR12 protein levels in human LVs and association of MI associated variant with echocardiographic parameters.

Representative Western blots are shown. A. WDR12 total protein and B. WDR12 nuclear protein levels in patients with cardiomyopathy and donor control hearts. Bands were detected from the same gel. C. WDR12 protein levels relative to donor control hearts. The results are expressed as mean±SEM. *P<0.05 versus control group (Student t test). D. Left atrial area/BSA (cm²/m²) in carriers of the previously myocardial infarction associated rs6725887 WDR12 genotypes and E. odds ratio (95% confidence intervals) for diastolic dysfunction (pseudonormalization + restrictive versus normal + relaxation impairment) in carriers of the previously myocardial infarction associated rs6725887 WDR12 genotypes.

https://doi.org/10.1371/journal.pone.0124907.g015

MI Associated WDR12 Allele Associates with Diastolic Dysfunction in Humans

In a crude additive model, the MI associated allele of rs6725887 (minor allele) was significantly associated with larger left atrial area/BSA (β-coefficient±SEM)(0.32±0.12, P = 0.006), which remained significant after age and sex adjustment (0.28±0.12, P = 0.016)(Fig 15D). Presence of significant diastolic dysfunction (pseudonormalization or restrictive pattern) was significantly associated with the MI associated allele of rs6725887 in crude additive model (odds ratio, 95% confidence interval) (1.42, 1.10‒1.83, P = 0.007) as well as after age and sex adjustment (1.38, 1.07‒1.79, P = 0.013)(Fig 15E). The association remained significant after additional adjustment for history of myocardial infarction (1.41, 1.09–1.82, P = 0.009). In contrast, there was no significant association between rs6725887 and variation of systolic function (LVEF) (P = 0.616).