The heart is the first organ to form and function in the embryo, and all subsequent events in the life of the organism depend on the heart’s ability to match its output with the organism’s demands for oxygen and nutrients. In our past research (the work was started by Dr. Igor Kostetskii in collaboration with Dr Glenn Radice, CRDF grant) we showed that cells adhesion, namely N-cadherin-catenin complex has principle role for cardiogenesis. Cardiospecific conditional knockout of N-cadherin leads to embryos lethality because of heart development violation (Fig1). Knockout of N-cadherin cytoplasmic partners: β-catenin or αE-catenin in embryonic heart did not cause the embryo’s heart malformations. But we registered lethality in the case of β-catenin missing latter in embryos development and postnatal. We assume that in contrast to N-cadherin, β-catenin is quite important for heart development because of its signalling function. β-catenin is important transcriptional co-activator of canonical Wnt signalling (Fig 1).
Fig 1 Cardiac-specific deletion of N-cadherin gene leads to cardiomyocyte adhesion defect and embryonic lethality. Whole mount images of wildtype (a) and N-cadherin CKO (b) E10.5 embryos. Note the malformed heart and pericardial edema (arrow) in the mutant embryo. Histological analysis of wild-type (c, e) and N-cadherin CKO (d, f ) E10.5 embryos. Note the less compact myocardial cell layer (arrow) in the mutant embryo (f ) (Piven O. et all, 2011)
Canonical Wnt signaling is one of the main signaling involved in control of the cell cycle, proliferation, apoptosis, and differentiation in many tissues and organs, including the heart. Missing of β-catenin has been compensating by plakoglobin in cells adhesion maintaining and adherent junctions (AJs) forming. Plakoglobin (γ-catenin) is a close relative of β-catenin. In contrast to β-catenin, it can interact with both classical cadherins in AJs and desmosomal cadherins in desmosomes. In addition, in our experiments, we revealed the activation of canonical Wnt signalling in β-catenin haploinsufficient new born heart (Axin2, TCF4 and c-Fos genes were overexpressed). Interestingly, at the same samples we observed an elevated level of Plakoglobin expression. On our opinion Plakoglobin and β-catenin interacts in a complex manner in regulating cell fate and possibly also during cardiogenesis. This functional redundancy in canonical WNT signaling regulation is likely weak and/or not effective at later stages of cardiogenesis.
In our present research together with Dr. Cecilia Winata Lab we applying the ChIP-seq method represent a revolutionary approach to study heart development. We also aim to elucidate the signaling function of γ-catenin during cardiac development, as well as investigate the functional redundancy between β- and γ-catenin
Different studies have shown that several Wnt factors are induced after experimental myocardial infarction (MI) in various animal models, being involved in hypertrophy and cardiac wound healing following injury. But signalling function of β-catenin in regulation of heart adaptation to hemodynamic, hormonal or training stress as well as to the ageing and regeneration is far from understanding. Here with β-catenin CKO mice and isolated cardiomyocytes using we shown that missing of β-catenin attenuated the cardiomyocytes maturation and specification. As a result full ablation of β-catenin in cardiomyocytes is lethal, when β-catenin haploinsufficient cardiomyocytes still express the foetal or hypertrophic genes (ANP, BNP and β-MHC) at high level in adult mice. We observed that cardiospecific β-catenin deficiency attenuated the adult heart adaptation to extending training in mice and occur the Akt kinase and ERK ½ signalling up-regulation. Together with Dr Pawel Dobrzun we are analizing requirement of β-catenin in fatty acid metabolism regulation under the training and ageing (Western blots, TLC and gas-liquid chromatography).
Thus, we believe that β-catenin signalling function is necessary for heart maturation and cardiomyocytes specification in new born heart as well as for effective adaptation of adult heart for training.
With BATGal transgenic mice using we have analyses signalling function of canonical Wnt and β-catenin under chronicle hypertension. Mice were implanted with mini-osmotic pumps (Aztech) containing either saline or AngII for 3, 7 or 14 days (Fig 2). In result we (together with Dr. Mathew Weller) observed the Wnt signalling activation after 3 days of AngII treatment, as well as b-catenin target genes and foetal genes up-regulation. In summary, our data showed that Wnt/b-catenin signaling is significantly increased during the early phase but is subsequently downregulated at later time points during development of pathological hypertrophy.
Fig 2. β-catenin signaling activated under hypertrophy stimulus (AngII an LiCl). With BatGal mice we observed positive β-galactosiase staining of cardiomyocytes at 3 day of mice treatment (arrow). With qPCR we also registered TCF4 gene upregulation in treated mice compare to control.
α-E-catenin mostly known for its structure function in cell-cell contact maintaining as component of AJs. During last decade it was shown that α-catenin modulates activity of Hippo-, Wnt-, Hedgehog- and NF-kB signaling pathways But signaling function of α-E-catenin in heart is far from being clear. That’s why in our present research we have addressed to requirement of α-E-catenin in signaling regulation of adult heart remodeling.
So, we generated the embryonic cardiomyocyte-specific deletion of α-E-catenin (αMHC-Cre). In our experiment we observed lethality of adult homozygous and heterozygous mice with α-E-catenin conditional knockout. We registered the lower average survival rate of mutant animal (Fig 3).
Fig 3 α-E-catenin ablation in heart leads to lower average survival rate of mutant animal. For animals with heterozygous deletion of α-E-catenin it was 38 ± 2 weeks, for animals with homozygous deletion of α-E-catenin – 36 ± 3 weeks, and for controls – 66 ± 4 weeks. The maximum life span for animals with partial loss of α-E-catenin was 48 weeks, while for animals with complete loss of studied gene – only 44 weeks (a); dramatic heart tissue fibrosis in α-E-catenin flox/flox mice (b) (Balatskii et all 2016)
It was found that α-E-catenin ablation in heart leads to hypertrophy in both mutant groups. We registered the higher ratio of hypertrophic index in haploinsufficient mice and mice with full deletion of studied gene compared to control. Also the level of hypertrophic genes ANP and β-MHC were significantly higher in mutant mice. The dramatic heart tissue fibrosis was observed in both mutant groups of mice with van Gieson staining (Fig 3). All these observations together evidence the critical function of α-E-catenin for adult heart development and remodeling.