Muscleblind-like 1 is required for normal heart valve development in vivo

Coram et al. BMC Developmental Biology (2015) 15:36 DOI 10.1186/s12861-015-0087-4 RESEARCH ARTICLE Open Access Muscleblind-like 1 is required for normal heart valve development in vivo Ryan J. Coram1,4, Samantha J. Stillwagon1,5, Anuradha Guggilam1, Michael W. Jenkins2, Maurice S. Swanson3 and Andrea N. Ladd1* Abstract Background: Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor β (TGFβ) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFβ signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1ΔE3/ΔE3 mice, which are null for MBNL1 protein. Methods: Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFβ signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. Results: As in chicken, the loss of MBNL1 promotes precocious TGFβ signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1ΔE3/ΔE3 mice exhibit valve dysmorphia with elevated TGFβ signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1ΔE3/ΔE3 mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy. Conclusions: Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFβ signaling, and is required for normal valve morphogenesis and homeostasis in vivo. Keywords: Muscleblind-like 1, Transforming growth factor β, Epithelial-mesenchymal transition, Endocardial cushions, Heart valves, Mouse Background Valve defects are among the most common congenital heart defects, and are associated with significant health problems and mortality throughout life [1]. Their exact incidence is unknown because many valve defects are not diagnosed until adulthood [2]. Aortic valve defects alone occur in 1-2 % of the population [3]. The prevalence of adults with valvular heart disease is increasing, due to improved surgical treatments and increases in lifespan that may be accompanied by valve degeneration [4]. Congenital valve defects and adult valve disease may * Correspondence: 1 Department of Cellular & Molecular Medicine, Lerner Research Institute, 9500 Euclid Ave. NC10, Cleveland Clinic, Cleveland, OH 44195, USA Full list of author information is available at the end of the article not be distinct, since structural or biomechanical abnormalities in the valves may be present but not readily appreciated at birth. The heart valves form by remodeling embryonic structures called endocardial cushions. Outgrowth of the cushions occurs via localized expansion of extracellular matrix (ECM) between the endocardium and myocardium in the atrioventricular canal (AVC) and outflow tract (OFT), after which the cushions are cellularized by invasion of a subpopulation of endocardial cells undergoing epithelial-mesenchymal transition (EMT) [5]. At post-EMT stages, this cushion mesenchyme differentiates into valve interstitial cells, specialized fibroblasts that produce the highly stratified matrix of the mature valves [2, 3]. The mitral and tricuspid valves form from the AVC © 2015 Coram et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Coram et al. BMC Developmental Biology (2015) 15:36 endocardial cushions, while the aortic and pulmonary valves arise from the OFT cushions [6]. Transforming growth factor β (TGFβ) signaling induces endocardial cushion EMT in both birds and mammals, although the roles of specific TGFβ paralogs differ [7–9]. In chick, TGFβ2 is required for activation of the endocardial cells, in which cell polarity and cell-cell contacts are lost, and TGFβ3 is required for invasion of the transformed mesenchymal cells into the cushion matrix [8, 9]. TGFβ1 is not expressed in the developing cushions. Both TGFβ2 and TGFβ3 are produced in the myocardium prior to the onset of EMT, and TGFβ3 is found in the endocardium during stages of active EMT [8, 10, 11]. Autocrine production of TGFβ3 in the cushion endocardium is essential for induction of invasive mesenchyme [11, 12]. In mouse, TGFβ3 is not expressed in the heart until E11.5, well after EMT is underway, and is restricted to the mesenchymal cells of the cushions [13]. No cardiac malformations have been found in Tgfb3-null mice [14, 15]. TGFβ2 has an expression pattern in the mouse heart that is similar to that of TGFβ3 in chick, and inhibition of TGFβ2 blocks invasion in mouse AVC explants [13, 16]. Tgfb2-null mice exhibit impaired EMT, hypocellular cushions, and a variety of valve and septal defects [7, 17, 18]. Thus, in mouse TGFβ2 plays a role orthologous to that of chick TGFβ3. TGFβ1 is also expressed in mouse AVC endocardium, and although it is not essential for endocardial cushion EMT in vivo, Tgfb1-null mice have disorganized valves [19]. We previously identified a role for muscleblind-like 1 (MBNL1) in the regulation of TGFβ-dependent EMT in the endocardial cushions in chick [20, 21]. MBNL1 is a member of the MBNL family of RNA binding proteins that regulate pre-mRNA alternative splicing, alternative polyadenylation, microRNA biogenesis, mRNA stability and localization [22–25]. We previously reported that in the embryonic chicken heart MBNL1 transcripts are found in the atrial and ventricular myocardium, and in the endocardium of the AVC and OFT endocardial cushions during EMT and immediately prior to its onset [20, 21]. We demonstrated that knockdown of MBNL1 in chick AVC or OFT explants promotes invasive mesenchyme formati (...truncated)