Palchevska O. The peculiarities of postnatal myocardium development with b-catenin gene heterozygous deletion

The study presented in this thesis is devoted to the research of the influence of b-catenin gene haploinsufficiency on the postnatal heart maturation and development as well as heart adaptation to prolonged physical training and hypertrophy. The b-catenin protein is well known as a main mediator of canonical Wnt/b-catenin signaling pathway on one hand and as a component of adherent junction on the other. The experimental evidence suggests the level of this protein to be critically important for the heart embryogenesis and development. So literature data concerning Wnt pathway role for postnatal heart function are quite controversial. The conditional knock-out animals were used. The gene expression experiments were performed using qPCR. The active protein forms expression was determined using Western blotting. The paraffin sections were stained with H&E and Masson's trichrome staining. The swimming test was adapted to model physical training. The primary cultures of cardiomyocytes isolated from transgenic animals were used for physiological activity measurements (MTT-test) and for cytological analysis. The primary cell cultures were stained with H&E staining. The statistical analysis of literature data (meta-analysis) was performed using linear regression and ANOVA. The effect size (g) was estimated using Eta squared. The experimental data statistical significance was also estimated using ANOVA. Using the transgenic b-catenin conditional knock-out animals the gene deficit was shown to provoke the adult heart maturation delay though at the same time the upregulation of hypertrophic genes was registered. The hypertrophic genes (known also as fetal) are associated with heart growth. The upregulation of fetal genes expression (ANP, BNP and b-MHC) was registered in the b-catenin haploinsufficient animal myocardium at the age of 1 and 3 months comparing to control animals. The downregulation of fetal genes ANP and BNP was also revealed in the myocardium with heterozygous deletion of b-catenin gene in animals at the age of 6 months comparing to the control ones, though the expression of b-MHC gene was still higher in mutants myocardium. The expression of TCF4 gene was shown to be higher in the myocardium of b-catenin haploinsufficient animals at all studied age points comparing to corresponding control. These observations point to the downregulation of canonical Wnt pathway in heart muscle under the condition studied. Also the c-fos, c-myc and cyclin D1 genes (known target genes of expression canonical Wnt pathway) expression supported of canonical Wnt pathway activation determined in previous experiment. The b-catenin gene haploinsufficiency condition and canonical Wnt signaling downregulation were shown to lead to primary cardiomyocytes metabolic activity decline and cell hypertrophy delay. The results presented in the thesis point to the important influence canonical Wnt pathway and b-catenin specifically have on the heart postnatal maturation and shift from fetal to adult genetic program. The canonical Wnt pathway downregulation causes perturbation in the both terminal differentiation and postnatal heart formation. The experiments with the myocardium adaptation to physical training under the condition of b-catenin gene heterozygous deletion revealed that signaling activity of b-catenin at least at basal level is necessary for normal adult myocardium adaptation. The downregulation of b-catenin signaling activity permits the heart adaptation only under the condition of canonical Wnt activation as well as increased phosphorylation of ERK1/2 and Akt that are components of MAPK and PI3K-mTOR-dependent signaling pathways. We suggest the compensatory effect between Wnt and other signaling pathways involved to hypertrophy development potentially supports the hypertrophy development when the canonical Wnt pathway activity is downregulated. The meta-analysis of literature data defined the panel of factors (namely, SERCA, different forms of actin, Axin2, c-myc, ANP and hypertrophic indices) as the most appropriate and statistically significant for hypertrophy development estimation. The results presented in the thesis expend current conception of b-catenin gene and canonical Wnt signaling pathway function in the formation, maturation and functioning of postnatal myocardium. The b-catenin signaling was shown to be critically important for postnatal heart development and for adult myocardium adaptation to physical training