Bicuspid aortic valve (BAV) is the most prevalent human congenital cardiac malformation, which predisposes to ascending aortic dilatation (AD). Disease predisposition probably results from a congenital defect leading to anomalies in both, the morphology of the valve and the histology of the ascending aorta. Altered hemodynamics caused by the abnormal valve morphology would accelerate aortic media degeneration. AD in BAV patients results from medial smooth muscle cell (SMC) apoptosis probably caused by defective cell‐matrix adhesion. Alterations of the TGFβ pathway seem to be central in disease initiation and progression, affecting extracellular matrix homeostasis. However, currently there are neither clear pathophysiological mechanisms nor effective biomarkers of disease predisposition or progression.The only spontaneous animal model of BAV disease consists of an inbred (T) strain of Syrian hamsters with a high (40%) incidence of BAV. Although AD has never been described in this model, animals of the T strain show significant SMC apoptosis and altered elastic lamina in the aortic media, irrespective of aortic valve morphology. We reasoned that analysing differentially expressed proteins in the aortic wall of animals with normal aortic valve (TAV) of the T strain compared to a control strain would inform about 1) the validity of the T strain as a model for BAV aortopathy predisposition; 2) the congenital origin of AD associated to BAV; 3) the proteins involved in AD etiology, independent of the effect of hemodynamics.To this aim, we compared the proteome of the ascending aorta of animals with TAV of the T strain (n=6) and a control outbred strain (n=6) by three independent experiments of quantitative proteomics (ESI‐Quadrupole‐Orbitrap LFQ). The analysis yielded expression data for over 1,954 proteins. Of these, 57 proteins (2.9%) showed concordant expression in at least two of the three experiments and were deregulated over 2.6‐fold.Fourteen of these 57 proteins (24.6%) were previously proposed as AD biomarkers in patients. After database search, the 57 selected proteins were found to significantly interact (p<0.05 following FDR adjustment) within 16 molecular pathways and 27 biological processes on the STRING website. Most of these pathways and processes were relevant to AD pathophysiology, of note are the TGFβ non‐canonical pathways (10/57; 17.5%; p=0.043), programmed cell death (14/57; 24.6%; p=3.5e‐4), cell adhesion (15/57; 26.3%; p=2.4e‐4), supramolecular fiber organization (9/57; 15.8%; p=3.8e‐4) and stress fiber assembly (2/57; 3.5%; p=0.037).These results were obtained comparing the proteomes of aortas of individuals with TAV from an affected and a control strain. Thus, genetic rather than hemodynamic factors explain the structural abnormalities of the ascending aorta in this model. We propose that the T strain is an appropriate model for AD predisposition, as it replicates the ethiophysiological processes observed in patients. Deep study of our ascending aorta proteome analysis may uncover new molecular ...

The abnormal origin of a coronary artery from the ascending aorta, above the sinotubular junction or high take‐off (HTO) is a rare congenital anomaly, which is associated with sudden cardiac death in approximately 1.5% of cases. HTO has been reported in 62.8% of mice from the C57Bl/6 (B6) strain, while it is absent in the Balb/c (BC) mouse strain. We have observed that the incidences of HTO in hybrid mice are intermediate between those of the parental animals (F1: 26.32%; F1xF1: 20%; F1xB6: 46.34%; F1xBC: 9.80%). In addition, we found that these incidences fit with a mode of inheritance consisting in the influence of a major causal dominant allele, present in the B6 strain, and a modifier dominant allele in the BC strain that reduces by half the incidence of HTO. In order to identify chromosomal regions including genes responsible for HTO development, we have carried out a genetic linkage study through the generation of a congenic strain, using microsatellite genetic markers. A region of 2,699,738 bp in chromosome 18 between the markers MIT‐49 and MIT‐106 was found statistically associated with HTO occurrence. This chromosomal region included a total of 9 genes (Smad2, Skor2, Ier3pi, Hdhd2, Katnal2, Pias2, St8sia5, Loxhd1, Rnf165). Among these, Smad2 was selected as a candidate gene, due to its multiple biological functions related to cardiovascular system development, specifically to aortic root formation and coronariogenesis. The comparative analysis of the Smad2 sequence between B6 and BC mouse strains revealed the presence of an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) located in the 5’ splicing zone of intron 10‐11. Sequence analysis predicted a possible error in Smad2 10‐11 intron splicing caused by the B6 missense variant (Smad2C>A). After genotyping a total of 104 B6xBC F2 to F5 hybrid individuals of the developing congenic strain, by means of PCR‐RFLP using Smad2 specific primers, we found a statistically significant (p<0.05) association between the allele Smad2C>A and HTO occurrence. We conclude that HTO is an inherited trait in mice, probably caused by a dominant allele and a dominant modifier allele. Our genotype‐phenotype association study points to the Smad2rs29725537:C>A allele as the main causal factor for HTO in mice. Further studies are required to 1) confirm the role of the Smad2C>A SNP in murine HTO development; 2) check whether human HTO development is also influenced by the Smad2C>A allele; and 3) investigate the possible involvement of the Smad2C>A allele in the development of additional traits, particularly those differentially expressed between mice of the B6 and BC strains.