JDP2 overexpression provokes cardiac dysfunction in mice

The transcriptional regulator JDP2 (Jun dimerization protein 2) has been identified as a prognostic marker for patients to develop heart failure after myocardial infarction. We now performed in vivo studies on JDP2-overexpressing mice, to clarify the impact of JDP2 on heart failure progression. Therefore, during birth up to the age of 4 weeks cardiac-specific JDP2 overexpression was prevented by doxycycline feeding in transgenic mice. Then, JDP2 overexpression was started. Already after 1 week, cardiac function, determined by echocardiography, decreased which was also resembled on the cardiomyocyte level. After 5 weeks blood pressure declined, ejection fraction and cardiac output was reduced and left ventricular dilatation developed. Heart weight/body weight, and mRNA expression of ANP, inflammatory marker genes, collagen and fibronectin increased. Collagen 1 protein expression increased, and fibrosis developed. As an additional sign of elevated extracellular matrix remodeling, matrix metalloproteinase 2 activity increased in JDP2 mice. Thus, JDP2 overexpression is deleterious to heart function in vivo. It can be concluded that JDP2 overexpression provokes cardiac dysfunction in adult mice that is accompanied by hypertrophy and fibrosis. Thus, induction of JDP2 is a maladaptive response contributing to heart failure development.

SCIEntIfIC RePoRts | (2018) 8:7647 | DOI: 10.1038/s41598-018-26052-w as homo-or hetero-dimers 3,4 , both of which are binding sites for AP-1. Upon dimerization of JDP2 with c-Jun, DNA binding is potentiated, but AP-1-mediated transcription is inhibited 3 . JDP2 inhibits transcription by multiple mechanisms which include competition for DNA binding sites of positive transcription factors 3 , competition with c-Jun N-terminal kinase phosphorylation 6,7 , promotion of nucleosome assembly activity 8 , and through direct recruitment of multiple members of histone deacetylases (HDACs) to cognate TRE-containing promoters 9 .
Cardiomyocytes isolated from JDP2-overexpressing transgenic mice revealed protection against the induction of hypertrophic growth and apoptosis 10 . These results imply a protective role of JDP2 in heart failure, and the induction of JDP2 after myocardial infarction may be an adaptive process. Another argument for the protective role of JDP2 is the recent finding, that knock out of JDP2 promotes cardiac hypertrophy and dysfunction in response to pressure overload in mice 11 . However, besides these positive effects, JDP2 also has detrimental effects in cardiomyocytes, since the contractile function of ventricular cardiomyocytes isolated from JDP2-overexpressing mice was impaired: While wildtype cardiomyocytes showed enhanced cell shortening as well as faster contraction and relaxation velocities with increasing amounts of the beta adrenoceptor agonist isoprenaline, JDP2-overexpressing cells failed to respond to β-adrenergic stimulation 10 . Due to these contradictory activities of JDP2 in cardiomyocytes, preventing processes of adverse cardiac remodeling, like hypertrophy or apoptosis, while simultaneously reducing their contractile capacity, it remains unclear, if JDP2 overexpression is protective or detrimental for the heart.
So far, the cardiac phenotype of JDP2 mice has been analysed in 4 week old mice. These studies revealed development of bi-atrial dilatation and defects in the conduction system 12 . No overt ventricular heart disease was detected. In histological sections of JDP2-overexpressing hearts, hypertrophic cardiomyocytes were detected in atria, but not in ventricles. However, as heart failure predominantly occurs in the elderly patient, we now determined the role of enhanced JDP2 expression in adult mice hearts. Since the expression of JDP2 in transgenic mice is under the control of a tetracycline-regulated α-MHC promoter, cardiac expression of JDP2 could be repressed by doxycycline feeding during juvenile development. In 4-week-old animals, JDP2 overexpression was started, and the effect of JDP2 on the cardiac phenotype and function was determined one and five weeks after JDP2 overexpression in adult mice.

Results
Cardiac function under JDP2 overexpression. Before analysing the phenotype of transgenic JDP2 mice, overexpression of JDP2 in absence of doxycycline feeding was evaluated in atria and ventricles by real time RT-PCR. In the absence of doxycycline JDP2 mRNA expression increased in atria 3.6-fold within one week, and 7.2-fold after five weeks (n = 6, p ≤ 0.05 vs. WT mice). In ventricles JDP mRNA was elevated 22.8-times within one week, and 10-fold within five weeks in absence of doxycycline feeding (n = 8, p ≤ 0.05 vs. WT mice). Furthermore, JDP2 protein expression was detectable in hearts of transgenic animals only (Fig. 1B). Experimental study design. JDP2-overexpressing mice were fed for four weeks with doxycycline (Dox). Then Dox-feeding was stopped and JDP2 was overexpressed for one or five weeks. Parameters related to cardiac performance were analysed. (B) Evaluation of JDP2 overexpression. mRNA expressions of JDP2 were evaluated. Data are means ± SD. *Differences from WT-animals with p ≤ 0.05, n = 8. Furthermore, JDP2 protein from 5 weeks overexpressing hearts was detected in western blots by HA antibodies. Vinculin was used as loading control.
SCIEntIfIC RePoRts | (2018) 8:7647 | DOI:10.1038/s41598-018-26052-w Next, we determined the influence of JDP2 overexpression on blood pressure. After one week of JDP2 overexpression blood pressure in WT and JDP2 mice were almost identical. After 5 weeks a significant drop in blood pressure was observed in JDP2 mice when compared to age-matched WT mice (129 ± 16 in WT vs. 110 ± 24 mmHg in JDP2 mice (n = 15 in each group, p ≤ 0.05) (Fig. 2). This decline in blood pressure may indicate a reduced ventricular pump function in JDP2 mice.

Short-term JDP2 overexpression impairs contractile function of isolated cardiomyocytes. As
we have previously demonstrated, life-long JDP2 overexpression reduces contractile performance in isolated cardiomyocytes 13 . This could explain the impaired cardiac function that we observe here in mice overexpressing JDP2 for five weeks. However, we could not exclude that during lifelong overexpression other secondary effects provoked contractile impairments in isolated cardiomyocytes. Therefore, we conducted additional measurements on mice that overexpressed JDP2 for only one week, as these mice already presented reduced cardiac function in echocardiography ( Table 2).
Cardiac apoptosis, hypertrophy, fibrosis, and inflammation under JDP2 overexpression. In order to analyse, if the functional impairment goes along with processes of apoptosis or hypertrophy, we performed TUNEL assay and determined heart to body weight ratio in mice overexpressing JDP2 for five weeks. TUNEL assay revealed a slight, but non-significant increase in apoptotic cells in JDP2 mice: 0.23 ± 0.10 TUNEL-positive cells per field were found in WT mice vs. 0.38 ± 0.11 in mice overexpressing JDP2 for 5 weeks (n = 5) (Fig. 5). An increased susceptibility to apoptosis was reflected in the enhanced expression of bax mRNA (2-times higher in JDP2 mice vs. WT, n = 8, p ≤ 0.05) and a decrease in bcl2 mRNA (0.2-times lower in JDP2 mice vs. WT, n = 8, p ≤ 0.05). Also on the protein level an increase in Bax expression to 198 ± 38% (n = 8, p ≤ 0.05 vs. WT) and a decrease in Bcl2 expression to 13 ± 4% (n = 8, p ≤ 0.05 vs. WT) was observed.
In order to obtain indications of altered signaling pathways in JDP2 mice, phospho-kinase array was performed. Interestingly, only a small number of proteins showed altered phosphorylation patterns. Out of the 43 analysed proteins, PDGF-receptor β was the only protein with a significant increase in phosphorylation to 129.6 ± 16.2% in JDP2 mice (n = 6, p ≤ 0.05 vs. WT). This indicates activation of PDGF-signaling, which is a central mediator of tissue fibrosis 14 .
To determine if inflammatory processes occur in JDP2 mice, mRNA expression of inflammatory marker genes was evaluated by real time RT-PCR. In mice overexpressing JDP2 for five weeks TNFα mRNA increased 2.3-fold and interleukin1β mRNA (IL1β) 2.1-fold (p ≤ 0.05 vs. WT, n = 8).

Discussion
The main finding of this study is that JDP2 overexpression in adult mice hearts is detrimental for heart function. Development of myocardial hypertrophy, fibrosis and cardiac dysfunction could be observed under JDP2 overexpression. Previous descriptions of heart-specific overexpression of JDP2 in transgenic mice showed atrial hypertrophy and defects in the conduction system, but no overt ventricular phenotype was detected 12 . These studies were performed in 4 week old mice under continuous JDP2 overexpression. In our study, we now initiated JDP2  . Apoptosis and Hypertrophy increases in JDP2 mice. Hearts were extracted from mice five weeks after start of JDP2 overexpression and from age matched WT animals. Apoptosis was detected by TUNELassay (n = 5). As hypertrophic parameter HW to BW ratio was determined (n = 15-22). Data are means ± SD. *Differences from WT animals with p ≤ 0.05.
SCIEntIfIC RePoRts | (2018) 8:7647 | DOI:10.1038/s41598-018-26052-w overexpression in the age of 4 weeks. Already one week after JDP2-overexpression impairment of ventricular function became evident, which was aggravated by extension of JDP2 overexpression to five weeks. Besides this impairment in ventricular function, atrial dilatation, ventricular hypertrophy, and fibrosis became evident in mice overexpressing JDP2 for 5 weeks. Main difference between our study and the one of Kehat et al. 12 , is the starting point of JDP2 overexpression, thereby indicating that JDP2 expression in juvenile mice is detrimental only in the atria, whereas in adult mice both atria and ventricles are affected. The occurrence of atrial dilatation in both studies (juevenile and adult mice), but induction of ventricular dysfunction only in adult mice, indicates that the atrial impairments are not the cause for development of ventricular dysfunction in adult animals. This is furthermore supported by our findings that ventricular dysfunction already develops in the first week of JDP2 overexpression. In addition, cardiomyocytes isolated after only one week of JDP2 overexpression, have a limited contraction capacity. This initial impairment of contractile function in ventricular cardiomyocytes may be the main reason for the reduced ventricular function of JDP2 mice in vivo. This conclusion is also supported by our own previous findings in isolated cardiomyocytes of JDP2 overexpressing mice, where we have shown that induction of JDP2 after birth impairs contractile function of cardiomyocytes within 7 weeks 10 . In the same study protective effects of JDP2 overexpression against induction of apoptosis or hypertrophy in isolated cardiomyocytes were found. However, no in vivo analysis on heart function of JDP2 overexpressing mice was performed at that time. This was made up for in the present study, revealing ventricular dysfunction in JDP2 mice already within one week of JDP2 overexpression. Combining the results of both studies, it can be assumed that the contractile dysfunction of cardiomyocytes plays a prevailing role in the phenotype in JDP2 mice and provokes ventricular dysfunction in vivo. The protective effects against hypertrophy and apoptosis which were found in isolated cardiomyocytes 10 appear to play a subordinate role that does not work anymore under the long lasting ventricular dysfunction.
Moreover, mice overexpressing the JDP2 homolog ATF3 also develop cardiac hypertrophy and dysfunction in absence of atrial defects 15 . In ATF3 mice, cardiomyocyte-macrophage cross talk seems to play a major role in the adverse cardiac effects of ATF3 16 . In our study we found induction of inflammatory marker genes in JDP2-overexpressing mice. Therefore, the activated immune system may contribute to heart failure progression in JDP2 mice.
Factors that are increased during JDP2 overexpression are TNFα and IL1β. Both of these cytokines can upregulate and activate MMPs that are responsible for collagen degradation and subsequent matrix remodeling 17 . IL1β is also a predominant factor for collagen deposition in cardiac remodeling. It is not only released from cardiomyocytes but also from macrophages and endothelial cells resulting in a cytokine burst that initiates a vicious cycle of stress contributing to adverse cardiac remodeling. Also in our model, we identified activation of MMP2,  18 . Another sign of ongoing fibrotic processes in JDP2 mice is the activation of PGFRbeta, since PDGF signaling is one of the central mediators in organ fibrosis 14 .
Interestingly, in our phospho-kinase array analysis PDGFRbeta was the only protein out of 43 that showed enhanced phosphorylation and thus activation in JDP2 mice. However, as we determined phospho-kinase levels at one time point only, we cannot exclude that other kinases were activated at earlier time points, and may also contribute to development of ventricular dysfunction.
Besides extracellular matrix remodeling and fibrosis induction, suppression of bcl2 expression was observed, and a trend to increased apoptotic levels. This potential loss of cardiomyocytes may additionally contribute to the reduced cardiac function. This result is consistent with the finding that the lack of JDP2 expression in neutrophils resulted in impaired apoptosis due to enhancement of bcl2 expression 19 .
Quite puzzling to our findings of impaired cardiac function under JDP2 overexpression is the fact, that knock out of JDP2 also provokes cardiac dysfunction in response to pressure overload 11 . However, these findings might mirror the divergent actions of JDP2: On the transcriptional control of gene expression JDP2 acts as specific AP-1 inhibitor but also as modulator of chromatin remodeling. And on the cellular level, JDP2 promotes contractile dysfunction but also impairs hypertrophy or apoptosis in cardiomyocytes 10 . In the study of Kalfon et al. 11 JDP2 knock out mice basically presents normal cardiac function. Only after aortic constriction, stronger cardiac impairments became evident in JDP2 knock out mice compared to WT animals. Under aortic constriction, the loss of anti-hypertrophic and anti-apoptotic actions of JDP2 might be missing in order to prevent pressure overload induced damage. In contrast to JDP2 knock out, JDP2 overexpressing mice develop cardiac dysfunction in absence of any additional pathological stimulus. In this situation the leading role of JDP2 is the impairment of contractile function.
Interestingly, in humans enhanced expression of JDP2 after myocardial infarction correlated with HF progression 2 . This indicates that under this situation, the unfavorable function of JDP2 prevails and contributes to heart failure progression. However, as Maciejak et al. 2 analysed JDP2 expression in PBMCs and we used cardiac-specific JDP2 overexpression, it remains to be determined, if JDP2 expression is increased also in cardiomyocytes after myocardial infarction to directly interfere with heart function in this setting.
In conclusion, the main finding of this study is that JDP2 overexpression in mice results in premature heart failure development. Thus, the promising protective effects against induction of hypertrophy and apoptosis, which were found in ventricular cardiomyocytes isolated from JDP2-overexpressing mice, could not be established in vivo. In contrast, JDP2 even has a detrimental character for heart function.

Materials and Methods
The  with Dox blocks the interaction between the transactivator and the promoter of the JDP2 gene, thereby preventing JDP2 overexpression. To suppress JDP2 overexpression during embryonic and juvenile development of mice, breeding pairs and newborn mice were fed with Dox until four weeks after birth. Then mice were fed with Altromin-standard diet for upto 5 weeks resulting in JDP2 overexpression (Fig. 1). Genotypes of JDP2 mice were tTA/tetO. As control, littermates of transgenic mice, which did not overexpress JDP2, (wild types, WT) were used. Thus, the WT group included the following genotypes 0/0, tTa/0 and tetO/0. WT mice received Dox-diet for the same times as the JDP2 mice.
Blood pressure determination. Blood pressure was measured in conscious mice using a blood pressure monitor from TSE Systems GmbH (Bad Homburg, Germany). Mice were set in a restrainer (60 mm diameter) which was placed on a 35 °C warm heating plate in order to ensure sufficient blood flow in the tail. A tail-cuff was placed to occlude blood flow and a volume pressure sensor probe was placed distally to measure blood pressure and heart rate.
Echocardiography. Cardiac function was assessed by transthoracic echocardiographic examination.
Echocardiography was performed as described previously 20 . Mice were anesthetized with isoflurane (5% for induction and 2% for maintaining anesthesia), the chest was shaved, and the animal was placed in supine position onto a heating pad. Two-dimensional and M-mode echocardiographic examinations were performed in accordance with the criteria of the American Society of Echocardiography with a Vivid 7 Cell isolation and culture. Mice were anaesthetized by isofluran inhalation. After cervical dislocation hearts were extracted, and retrograde-perfused in a Langendorff apparatus with a collagenase-containing calcium-free buffer equilibrated at 37 °C, pH 7.4. After separation of cardiomyocytes from other cardiac cells by centrifugation, medium was readjusted to a physiological calcium concentration and suspended in basal culture medium. Cardiomyocytes were then plated on laminin coated culture dishes. After 2 hours medium was changed and cells could be stimulated. The basal culture medium (CTT) was modified medium 199 including Earl's salts, 2 mM L-carnitine, 5 mol/L taurine, 100000 IU/L penicillin, 100 mg streptomycin and 10 μmol/L cytosine-beta-D-arabinofuranoside.
Cell contraction. Cell shortening was analysed as described previously 13 . Briefly, cardiomyocytes were stimulated at 2 Hz for 1 min at room temperature for analysis of basal contraction or under incubation with 10 nM isoprenaline. Analysis of cell contraction was performed using cell-edge detection system. Cells were stimulated via two AgCl electrodes with biphasic electrical stimuli composed of two equal but opposite rectangular 50 V stimuli of 5 ms duration. Only rod-shaped cells that contracted regularly during the whole time of measurement were used. Every 15 s cell shortening, contraction and relaxation velocities were measured using a line camera. The mean of four measurements per cell was used as average value of each individual cardiomyocyte. Cell shortening data were normalized to the individual diastolic cell length (dL/L %).

Isolation of Hearts.
Mice were committed to euthanasia by isoflurane inhalation (5% isoflurane) and cervical dislocation. Hearts were directly excised and blood was washed out with ice-cold 0.9% NaCl. Afterwards tissue was either shock frozen in liquid nitrogen and stored at −80 °C or frozen tissue sections were made.
Real time RT-PCR. Total RNA from left ventricles was extracted with Trizol (Invitrogen) as described by the manufacturer. This was followed by DNAse treatment and reverse transcription with QuantiTect Reverse Transcription Kit from Qiagen. For each assayed gene, annealing temperature and the number of cycles resulting in a linear amplification range were tested. Real time RT-PCR was performed in an automated thermal cycler and was detected with the Biorad detection system (Biorad) using SYBR Green fluorescence for quantification. The calculations of the results were carried out according to the 2 −ΔΔCt methods as described 22 . Gene expression was related to hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping gene. Primer sequences are listed in Table 2.
Phospho-kinase array. For detemination of kinase phosphorylation patterns in cardiac protein extracts we used Human phospho-kinase array kit form R&D systems. This kit enables detection of relative levels of phosphorylation of 43 kinase phosphorylation sites and 2 related total proteins. The array was used as described by the company.
Frozen Tissue sections. Whole ventricles are embedded in "Tissue Freezing" (R. Langenbrinck) on Cryomold bowls (Weckert Labortechnik). Then sample are snap-frozen in 2-Methylbutane-isopentan (Fluka). Frozen tissues are cut into slices and used for determination of fibrosis or apoptosis (see below).
TUNEL Assay. After digestion of frozen tissue sections with proteinase K, samples were labeled with biotin-labeled dUTP using terminal transferase. Apoptotic nuclei became visible by streptavidin texas red staining.
Fibrosis Assay. Frozen tissue sections were stained with hematoxylin-eosin to detect muscle tissue in red and azan dye to detect collagen in blue. Micrographs were taken, and fibrotic areas were determined using Image J software.
Zymography Assay. Gelatinolytic activities of MMP-2 were examined as previously described in detail 23 .
Briefly, 8% polyacrylamide gels, co-polymerized with gelatin (2 mg/ml) were loaded with 50 µg of protein. After electrophoresis, gels were washed with renaturation buffer (containing 2.5% Triton X-100), and then incubated in development buffer to eliminate Triton-X-100. Gels were stained with 0.05% Coomassie brilliant blue, and gelatinolytic activities were detected as transparent bands against the dark-blue background. Band intensities were quantified (Quantity One software, Bio-Rad,Hercules, CA) and expressed in arbitrary units.
Statistics. Data are given as mean ± standard deviation from n different animals. Statistical comparisons were performed by ANOVA (One-Way Analysis of Variance) and Student-Newman-Keuls test or student-T-test. A p-value of less than 0.05 was considered statistically significant.
Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.