Embryonically induced β-catenin haploinsufficiency attenuates postnatal heart development and causes violation of foetal genes program

The β-catenin role in myocardium remodeling and hypertrophy development is the subject of numerous and controversial investigations. Aim. To investigate the significance of cardiac ablation of β-catenin for heart development using conditional knockout approach. Methods. Standard histological techniques (HEand MT-staining) and quantitative RT-PCR were used. Results. Our data demonstrate that β-catenin haploinsufficiency in heart provokes upregulation of foetal genes program without visible morphological abnormalities comparing to control groups of animals of the same age. Conclusions. Our data demonstrate that experimental conditions in this study provoke the delay in the development and growth of adult heart without visible morphological abnormalities.

Introduction.The adult heart is a dynamic organ capable of significant remodeling and hypertrophic growth for adaptation to altered workloads or injury.Hemodynamic stress or neuroendocrine signaling associated with myocardial infarction, hypertension, aortic stenosis, and valvular dysfunction evoke a pathologic remodeling response through the activation of intracellular signaling pathways and transcriptional mediators in cardiac myocytes [1].Activation of these molecular pathways may initially augment cardiac output (adaptive hypertrophy), however, prolonged hypertrophy (pathological or maladaptive hypertrophy) leads to heart failure and sudden cardiac death (SCD).Whilst there have been major advances in the identification of genes and signaling pathways involved in mediating hypertrophy it is clear that due to the overall complexity of hypertro-phic remodeling further characterization of the underlying molecular mechanisms is needed.
One such signaling pathway that plays a major role in both heart development and normal heart homeostasis is the Wnt/b-catenin pathway [2][3][4][5][6][7].Beta-catenin is an armadillo protein family member and has dual function forming an essential component of the adherent junction mediating cell-cell contact and acting as a transcriptional co-activator of the T-cell factor/lymphoid enhancer factor (TCF/LEF) complex [8][9][10].During cardiogenesis, Wnt/b-catenin activity displays a biphasic role as it appears to be required for cardiac progenitor cell (CPC) specification and expansion during early development but needs to be fine-tuned at later stages to allow terminal differentiation [11][12][13].
Conditional inactivation of b-catenin in endothelial cells prevents endothelial-mesenchymal transformation during cardiac development [4].A number of experimen-INDUCED b-CATENIN HAPLOINSUFFICIENCY ATTENUATES POSTNATAL HEART DEVELOPMENT tal works demonstrated that loss of b-catenin at early stages of heart formation using different tissue-specific promoters led to multiple embryonic heart malformation and had lethal effect [14][15][16].Meanwhile, conditional knockout of b-catenin after first and second heart field formation does not lead to early embryonic death, possibly due to functional redundancy between b-catenin and plakoglobin (g-catenin), but leads to lethality of late embryos and newborn mice [17,18].Interestingly, g-catenin is a close relative of b-catenin [19] but in contrast to b-catenin it can interact with both classical cadherins in AJs and desmosomal cadherins in desmosomes.It was shown that ablation of b-catenin in the adult heart does not result in lethality or other morphological heart malformations, presumably due to compensation by plakoglobin [20].Consistently, it was shown that cardiac-specific deletion of both g-catenin and b-catenin in adult heart leads to cardiomyopathy resulting in SCD [21].It has been demonstrated that activation of b-catenin is not only sufficient but also required to induce cardiomyocyte hypertrophy, and interestingly, the activation of b-catenin can occur independently of Wnt stimulation [22,23].The importance of b-catenin-dependent signaling for the development of cardiac hypertrophy has also been confirmed in vivo using mice with conditional cardiac-specific knockout of b-catenin [24].Cardiac-specific haploinsufficiency of b-catenin attenuates pressure-overload-induced cardiac hypertrophy after transverse aortic constriction (TAC) [18] while overexpression of a constitutively active form of b-catenin results in dilated cardiomyopathy [25].
In another study by Hahn et al. [26] the overexpression of a constitutively active form of b-catenin led to spontaneous hypertrophy of cardiomyocytes in culture.However, the role of b-catenin during heart reconstruction is still unclear with some investigations demonstrating opposing results.For example, it was reported that b-catenin downregulation was necessary for adaptive cardiac remodeling and cardiac hypertrophy development [27].
In this study we have addressed the role of b-catenin during postnatal heart development and remodeling.For this purpose we induced embryonic b-catenin haploinsufficiency in heart using Cre-mediated technique.We have analyzed the heart morphology and physiological hypertrophic remodeling of mice at different time points (1, 3 and 6 month) under b-catenin haploinsufficiency conditions and in normal animals in vivo.
Materials and methods.Generation of mutant mice.To generate cardiac-specific deletion of b-catenin b-cat flox/+ , a-myosin heavy chain (a-MHC)-Cre mice were mated with b-cat flox/flox mice.The mice with heterozygous floxed b-cat, a-MHC-Cre transgene were designated as mutants and were used for the analysis, the mice of all other genotypes served as controls.Alfa MHC-Cre transgene elicited recombination in cardiac muscles, but not other organs [28,29].Male mice were used in all experiments.
Homozygous conditional b-catenin flox/flox mice and a-MHC-Cre mice were kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).
Results and discussion.Embryonically induced bcatenin haploinsufficiency in heart did not lead to lethality but affected postnatal heart development.As we reported earlier [17] cardiospecific ablation of b-catenin at early embryonic stages leads to lethality at late gestation.In present study we focused on the analysis of the embryonical ablation of b-catenin on postnatal heart development.We studied how b-catenin haploinsufficiency condition is reflected on postnatal heart development and growth.We generated b-cat-CKO mice as described earlier [17].Mice were sacrificed at 1, 3 and 6 month of age.For evaluation of adult heart hypertrophy response the HW/BW index was calculated.We did not find any significant differences between HW/BW index of WT and b-catenin haploinsufficient mice at one month of age (1-month-old mice) (Fig. 1).When analyzing older mice (3 and 6 month of age) we observed some changes in mutant mice group as shown in Fig. 1.At three month of age b-catenin haploinsufficient mice had lower HW/BW ratio as compared to wild type.With ageing (6 month) we revealed only a tendency to increase the HW/BW ratio in mutant group (Fig. 1).
These data suggested that b-catenin haploinsufficiency provoked a delay in the development and growth of the adult heart (via the effect ANP and BNP on myocardium) probably with following hypertrophic responses development during ageing as a result of secondary compensatory effect.
Beta-catenin haploinsufficiency didn't lead to morphological malformations and heart tissue fibrosis.Despite the age-related changes in HW/BW ratio the heart tissue morphological analysis of wild type and b-catenin haploinsufficient mice did not reveal any difference (Fig. 2, see inset).HE stained heart sections of mice with genotype a-MHC-Cre + , b-catenin flox/wt looked normally: we have not observed any clearly marked tissue malformations in all analyzed groups (data not showed).Additionally we did not observe tissue fibrosis of control and b-catenin haploinsufficient hearts in any of analyzed age-ing groups (Fig. 2, see inset).
Probably, b-catenin haploinsufficiency in heart does not change tissue architecture in analyzed groups of animals due to the functional redundancy between b-catenin and plakoglobin, taking into consideration that plakoglobin is capable to maintain adherent junction.
Beta-catenin deficiency in adult heart led to upregulation of hypertrophy response genes in age-dependent manner.We have analyzed the level of hypertrophic response genes (ANP, BNP, b-MHC and a-MHC) expression in WT and experimental groups of mice at 1, 3 and 6 month of age.It is well-known that overexpression of ANP, BNP, b-MHC genes and a-MHC gene downregulation are associated with heart hypertrophic remodeling.Here we compared how b-catenin haploinsufficiency in heart affected these specific genes expression in different ageing groups.
Using qPCR we have revealed that hypertrophic genes programs were upregulated at 1 and 3 month of age (Fig. 3, 4) under b-catenin haploinsufficiency conditions comparing with control groups of animals of the same age.
Notably in both age groups of mutant mice the a-MHC gene expression was lower comparing with cont-   Analyzing the hypertrophic genes expression for six months old heart we revealed that expression of ANP and BNP genes was lower (Fig. 5) in b-catenin deficient mice comparing to control animals of the same age (Fig. 5) and to mice of 1 and 3 months old (Fig. 3, 4).
In contrast to previously studied age groups, in six month age mice the level of expression of a-MHC gene was higher under b-catenin haploinsufficiency conditions than in WT animals.But b-MHC expression level remained at elevated level similarly to previous age groups.
From our data we can conclude that cardiospecific induction of b-catenin haploinsufficiency in mice embryos leads to hypertrophy genes upregulation at 1 and 3 month of age with following downregulation of some of them (ANP and BNP) in older mice (6 month).
We would like to note that in wild type mice the expression of b-MHC is turned off shortly after birth, expression of a-MHC gene in the ventricular myocardium is upregulated and this protein isoform remains to be predominant in adults [28,31].With aging and during cardiac hypertrophy and failure, a-MHC expression is downregulated [32][33][34].Thus, expression of a-MHC gene is altered in a variety of physiological and disease states.The fact that b-MHC gene expressed in all studied b-catenin haploinsufficient groups at higher level comparing with control mice is the evidence that typical healthy adult heart genes program is disrupted.
The signaling and structural functions of b-catenin in embryos and adult heart development are being studied by different groups of scientists.Meanwhile, the role of b-catenin during adult heart reconstruction (heart hypertrophy or heart adaptation to ageing and training) is still unclear, even more, some studies demonstrated controversial data.
In present work we generated mice with cardiospecific b-catenin deficiency and analyzed how this condition contributes to postnatal heart development and adult heart formation.Our data demonstrate that b-catenin haploinsufficiency in heart provokes the delay in the development and growth of the adult mice heart (at 3 month of age) but without any morphological abnormalities comparing with control groups of animals of the same age.On the other hand we observed that b-catenin haploinsufficiency leads to hypertrophic response genes upregulation at 1 and 3 month of age.These findings suggest that the reactivation of the foetal gene program is not always linked with hypertrophic growth.This observation is consistent with Dr. F. Li group results [18].These authors induced cardiospecific ablation of one b-catenin gene allele but in adult mice (3-4 month of age).As a result they observed that b-catenin haploinsufficiency attenuated heart hypertrophy but enhanced the foetal genes expression after TAC [18].Given this the survey of gene expression (genes of hypertrophic response as well as other cardiac Wnt-responsive In our experiment we also registered some foetal genes program upregulation in age-dependent manner.In 6-month-old animals we observed that ANP and BNP genes were downregulated but both band a-MHC genes were overexpressed comparing with control animals.Such processes are not typical for adult mature heart, and should be the sign of some abnormality in mutant mice especially given that young organism was growing actively.We can speculate that some compensatory effects were involved in ageing heart adaptation under b-catenin haploinsufficiency conditions.It was shown by another group of authors [21] that ablation of b-catenin and plakoglobin in adult heart leads to hyperthrophy and lethality.Authors made a suggestion that these effects are mediated by WNT/b-catenin-independent unspecified compensation which is supported by the evidence obtained with other models usage [7,35].
Noteworthy also is the hypothesis that although ANP and BNP are marker genes of hypertropic response widely used in clinics their function in cardiomyocytes under hypertrophic stimuli is rather compensatory [36,37].Their expression in cardiomyocytes is responsive to physical and/or chemical hypertrophic stimuli and contributes to numerous different physiological processes regulation.In particular they are known to decrease apoptosis and fibrosis as well as protein and DNA synthesis levels in cardiomyocytes developing hypertrophy.The upregulation of foetal genes in the b-catenin haploinsufficient cardiomyocytes comparing to control lit-

Fig. 1 .
Fig. 1.Analysis of control (1) and mutant (2) mice heart development of different ageing groups.Quantification of heart weight/body weight ratios of b-catenin flox/wt/Cre + and WT mice at 1, 3 and 6 months were performed.Note WT n ³ 30, CKO n ³ 15 in each group

Figure 2
Figure 2 to article by O. L. Palchevska et al.

Fig. 3 .
Fig. 3.The hypertrophic response genes expression in bcatenin haploinsufficient versus WT mice at 1 month of age.Each group consists of 2 mice