Abstract
Heart valve disease affects up to 30% of the population and has been shown to have origins during embryonic development. Valvulogenesis begins with formation of endocardial cushions in the atrioventricular canal and outflow tract regions. Subsequently, endocardial cushions remodel, elongate and progressively form mature valve structures composed of a highly organized connective tissue that provides the necessary biomechanical function throughout life. While endocardial cushion formation has been well studied, the processes required for valve remodeling are less well understood. The transcription factor Scleraxis (Scx) is detected in mouse valves from E15.5 during initial stages of remodeling, and expression remains high until birth when formation of the highly organized mature structure is complete. Heart valves from Scx-/- mice are abnormally thick and develop fibrotic phenotypes similar to human disease by juvenile stages. These phenotypes begin around E15.5 and are associated with defects in connective tissue organization and valve interstitial cell differentiation. In order to understand the etiology of this phenotype, we analyzed the transcriptome of remodeling valves isolated from E15.5 Scx-/- embryos using RNA-seq. From this, we have identified a profile of protein and non-protein mRNAs that are dependent on Scx function and using bioinformatics we can predict the molecular functions and biological processes affected by these genes. These include processes and functions associated with gene regulation (methyltransferase activity, DNA binding, Notch signaling), vitamin A metabolism (retinoic acid biosynthesis) and cellular development (cell morphology, cell assembly and organization). In addition, several mRNAs are affected by alternative splicing events in the absence of Scx, suggesting additional roles in post-transcriptional modification. In summary, our findings have identified transcriptome profiles from abnormal heart valves isolated from E15.5 Scx-/- embryos that could be used in the future to understand mechanisms of heart valve disease in the human population.
Highlights
Heart valves facilitate unidirectional blood flow during the cardiac cycle and this is largely achieved by highly organized layers of connective tissue that each offer distinct biomechanical properties to facilitate opening and closing of the valve leaflets or cusps [1,2]
Connective tissue homeostasis is mediated by valve interstitial cells (VICs), which in turn are regulated by a monolayer of valve endothelial cells (VECs) that overly the valve surface
Samples were subject to RNA sequencing (RNA-seq) using Illumina HiSeq 2000 following confirmation of a 1523.23 6 58.68 fold decrease in Exon 1 expression in Scx-/- samples compared to controls, as previously described [10]
Summary
Heart valves facilitate unidirectional blood flow during the cardiac cycle and this is largely achieved by highly organized layers of connective tissue that each offer distinct biomechanical properties to facilitate opening and closing of the valve leaflets or cusps [1,2]. Diseased or dysfunctional valves from pediatric and adult patients are characterized by VIC disarray, VEC denudation and disorganization of the connective tissue leading to biomechanical insufficiency [3,4]. There is no effective treatment for valve disease and over 50,000 valve replacement surgeries are performed in the United States each year [5]. Many of these procedures using bioprosthetic or mechanical valves fail due to structural or thrombotic-related problems and alternatives are needed [6]. Defects in embryonic valve development are responsible for congenital heart valve malformations, there is increasing evidence to suggest that they attribute to disease later in life [2]
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