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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Dev Dyn. 2010 Jul;239(7):2118–2127. doi: 10.1002/dvdy.22346

Expression Of The Familial Cardiac Valvular Dystrophy Gene, Filamin-A, During Heart Morphogenesis

RA Norris 1,★, R Moreno-Rodriguez 1, A Wessels 1, J Merot 2, P Bruneval 6, AH Chester 7, MH Yacoub 7, A Hagège 3, SA Slaugenhaupt 4, E Aikawa 8, JJ Schott 2, A Lardeux 2, BS Harris 1, LK Williams 1, A Richards 1, RA Levine 5,*, RR Markwald 1,*
PMCID: PMC2909582  NIHMSID: NIHMS213991  PMID: 20549728

Abstract

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

Keywords: Filamin, Cytoskeleton, Valve, Epicardial, Endothelium, Cardiac Morphogenesis, Development, Cushion, Mesenchyme, Mitral Valve Prolapse

Introduction

The four chambers of the heart are separated by valves that open to allow unidirectional blood flow. The mitral valve is located between the left atrium and the left ventricle, and the failure of the mitral leaflets to appose normally results in mitral valve prolapse (MVP, MIM 157700). Originally described in the 1960s, MVP is characterized by the systolic displacement or billowing of the mitral leaflets into the left atrium and is often accompanied by mitral regurgitation (MR) (Barlow and Bosman; 1966). MVP is the most prevalent of all mitral valve diseases, affecting as many as 2.4% of the population, and is the leading cause for mitral valve repair operations (Freed et al.; 2002a; Freed et al.; 2002b). Mitral leaflets in affected individuals exhibit excess tissue growth with myxomatous changes, characterized by altered collagen and elastin composition, proteoglycan accumulation, and disruption of the fibrous backbone. Many of the patients with MVP develop serious complications, including bacterial endocarditis, ruptured chordae tendineae, progressive mitral regurgitation, arrhythmias, and even sudden death (Devereux; 1989; Devereux et al.; 1989; Devereux et al.; 1986). Although the etiology of MVP is unknown, based on preliminary data shown below we hypothesize that MVP is a result of developmental defects during cardiac valvulogenesis, which over time result in clinical expression. If true, normal mature valves are dependent on integration of the molecular signals, cellular responses, and biomechanical stimuli that occur during cardiac morphogenesis.

The initial stages of valvulogenesis commence with the formation of atrioventricular (AV) and outflow cushions that function as primitive valves by blocking retrograde blood flow. The midline superior and inferior AV cushions, as well as the cushions of the distal outflow tract (truncus region) (OFT), form by a well-studied endothelial to mesenchymal transformation (EMT) (Bernanke and Markwald; 1982; DeRuiter et al.; 1997; Eisenberg and Markwald; 1995; Markwald et al.; 1977; Markwald et al.; 1975; Markwald and Smith; 1972; Mjaatvedt et al.; 1987; Mjaatvedt and Markwald; 1989). The fate of the cushions in the AV canal is to fuse and integrate with mesenchyme of the dorsal mesenchymal protrusion (DMP) and atrial cap to form an AV mesenchymal septal complex (Perez-Pomares et al.; 2002; Snarr et al.; 2008; Snarr et al.; 2007). This complex contributes to the development of two of the AV valves (aortic leaflet of the mitral valve and the septal leaflet of the tricuspid valve). The development of the other two AV leaflets (mural leaflets of the tricuspid and mitral valves) involves the formation of “lateral” cushions that are formed at the left and right side of the AV junction (de Lange et al.; 2004; Wessels et al.; 1996). Similarly, in the OFT, cushions composed of both EMT-derived mesenchyme and neural crest cells, integrate to form the aortic and pulmonic (semilunar) prevalvular leaflets. Whereas much is known about the EMT process that gives rise to the major/midline AV and OFT cushions, much less is currently known about the mechanisms driving post-EMT cushion remodeling and maturation of the definitive AV or OFT valves. Adding to the complexity of AV valve morphogenesis are the relatively unknown mechanisms by which the suspensory (tension) apparatus (annulus and tendinous cords) are formed. It is also generally understood that the mature mitral and tricuspid leaflets as well as the aortic and pulmonic valves develop a specific architecture with a trilaminar appearance of elastin, proteoglycans, and collagen I (Cole et al.; 1984; Hinton et al.; 2008; Hinton et al.; 2006; Lincoln et al.; 2004). The “post-EMT” regulatory mechanisms by which these matrix components and supporting structures are laid down, the timing of their deposition, and the role of the interstitial valve fibroblasts are poorly understood. Disruption of any of these morphogenetic processes during valve development would be expected to affect the overall structure and function of the mature valves. Our understanding of specific genes involved in valve maturation has been somewhat hampered by the lack of relevant clinical data implicating specific gene mutations as causal to mitral valve disease. However, four gene loci have been recently identified by our group as containing putative MVP genes; thus suggesting genetic heterogeneity of this disease ((Disse et al.; 1999; Freed et al.; 2003; Kyndt et al.; 1998; Nesta et al.; 2005)).

One of these loci contained the cytoskeletal actin-binding protein Filamin-A which was found by genetic approaches to harbor specific mutations that cause myxomatous valves in an X-linked form of familial cardiac valvular dystrophy (Kyndt et al.; 2007). Filamin-A is a member of the filamin group of proteins that consists of three members: filamin-A, filamin-B, and filamin-C. The Filamins are present as homo- or heterodimeric Y-shaped cytoplasmic proteins, with each main chain consisting of an actin-binding region at the amino terminus. The core of the protein consists of 24 highly homologous Ig-like repeats followed by a carboxyl integrin-binding domain (Popowicz et al.; 2006). The most widely studied function of these large proteins is their ability to cross-link cortical actin filaments into a dynamic three-dimensional structure (Feng and Walsh; 2004). Surprisingly, the filamin-A knock-out mouse exhibits very specific defects in post-EMT cardiac morphogenesis suggesting a crucial developmental role for this protein in regulating valve maturation (Feng et al.; 2006; Hart et al.; 2006). Additionally, humans with point mutations in the filamin-A gene progressively exhibit ‘non-syndromic’, myxomatous valve defects without other developmental abnormalities. The key question is whether the MVP phenotype is the result of the expression of filamin-A mutations during development. If so, this would suggest that valve degenerative diseases can have “roots” in development. This new information warrants a detailed examination of this gene/protein during valve morphogenesis. Thus, the temporal and spatial pattern of Filamin-A expression during cardiac development presented in this report will serve as essential information that will potentially lead to the identification and characterization of new and relevant molecular and cellular pathways driving valve morphogenesis and will facilitate the discovery of Filamin-A-dependent mechanisms that contribute to the pathogenesis of mitral valve prolapse.

Material and methods

Immunohistochemical Analysis of Filamin-A Protein Expression

Mouse embryos at embryonic day (E) 9.5, 11.5, 13.5 and isolated hearts from E17.5, neonatal (2 weeks) and adult (6 months) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Deparaffinized sections were rehydrated through a graded series of ethanols to phosphate buffered saline (PBS-Sigma, St. Louis, MO.). Sections were subjected to antigen unmasking (H-3300; Vector Laboratories, Burlingame, CA) and treated for 1 hr at room temperature with a blocking buffer of PBS (Sigma, St. Louis, MO) containing 5% normal goat serum, (NGS, Cappel, Malvern, PA), and incubated with both an affinity-purified rabbit anti-human Filamin-A monoclonal antibody (Epitomics, Inc) diluted to 1:250 and a 1:50 dilution of myocyte-specific mouse monoclonal MF20-c (Developmental Studies Hybridoma Bank, Iowa City, IA) in blocking buffer overnight at 4°C. Following primary antibody incubations, specimens were washed five times in PBS and incubated at room temperature with Alexa Fluor goat α-rabbit 488 and goat α-mouse 568 (Invitrogen, Eugene, OR) diluted 1:100 in PBS. Nuclei were stained with Hoechst dye (1:10,000) (Invitrogen) in PBS for 5 min prior to the final washes in PBS. All samples were cover-slipped using Dabco mounting medium (Sigma). Controls for immunohistochemistry included omission of the primary antibody and preabsorption of the primary antibody with the immunopeptide prior to addition on the section (data not shown). Images of immunostained sections were captured with a Leica DM IRB Microscope System (Leica Microsystems, Inc Exton, PA). Files were transferred to Adobe Photoshop for labeling and figure preparation. Additional staining methods are provided in the supplemental material.

Histochemical and Immunohistochemical Analysis of Human Valves

Human mitral valve leaflets were collected from explanted hearts, transported to the laboratory in Media 199 and subsequently fixed in 10% formalin for 24 hours, processed and embedded into wax blocks. Control human valves were collected from a male donor whose cause of death was given as an intracranial bleed. Myxomatous valves were obtained from a male with severe mitral degenerative valve disease (Barlow’s disease). Ethical permission for the collection of valve tissue was given by the Royal Brompton & Harefield NHS Trust Research Ethics Committee. All chemicals for performing the Movat’s stain were purchased from Electron Microscopy Sciences. The protocol was followed per manufacturer’s recommendations. Immunohistochemical stainings using an α-smooth muscle actin (α-SMA) specific antibody (1:500, Sigma) and Filamin-A (1:250, Epitomics) were performed as described above.

Results

Cardiac expression of filamin-A at E9.5 is confined to the endocardium and mesenchyme of the atrioventricular (AV) and outflow tract (OFT) cushions (Figure 1B,E,F). Expression is not detected within the myocardium. By E11.5, filamin-A is robustly expressed in all endothelial and mesenchymal cells of the AV and OFT cushions (Figure 2), in addition to the epicardial epithelium covering the ventricles and the AV sulcus. At this timepoint, trabecular endothelium also expresses filamin-A. By E13.5 the inferior and superior cushions have fused and become part of the forming atrioventricular septal complex (AVSC). Filamin-A continues to be expressed throughout the fused cushions, being present in both the endothelium and mesenchyme. At this E13.5 timepoint, the left and right lateral AV cushions have developed and express filamin-A throughout the endothelium and mesenchyme. Additionally, the left and right AV sulcus and trabecular endothelium (arrows) express filamin-A (Figure 3A–C). The aortic wall, the forming outflow tract septum, and epicardium all exhibit filamin-A expressing cells (Figure 3D–F).

Figure 1. Immunohistochemical Analysis of Filamin-A at E9.5.

Figure 1

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(A,D) H&E staining showing gross morphology of E9.5 mouse embryo. (B) Low magnification of Filamin-A (Green) and MF20 (Myocardium-Red) showing widespread expression of Filamin-A in the mouse embryo with high levels seen in the brachial arches (Br), developing facial prominences, outflow tract (OFT) and atrioventricular (AV) regions. (C) Higher magnification of Filamin-A of boxed area in B, showing robust expression in the proepicardial organ (PEO) and epicardial cells (epi--arrows) emanating from this tissue. (E,F) Higher magnification of Filamin-A and MF20 expression in the developing brachial arches (Br), OFT, and atrioventricular canal showing robust expression in the endothelium of the OFT and AV cushions and in transformed mesenchyme (arrow heads). Filamin-A expression is absent in the myocardium (MYO). Nuclei—blue (Hoescht stain). Scale bars: A, B = 200 μm, C–F: 25 μm.

Figure 2. Localization of Filamin-A in the E11.5 mouse heart.

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(A) H&E showing gross morphology. (B) Filamin-A (green) is intensely expressed in the superior and inferior atrioventricular cushions (AV), outflow tract cushions (OFT), epicardium (arrow) and forming trabeculae endothelium in addition to forming blood vessels (BV). (C) Higher magnification of boxed area in B showing intense Filamin-A expression in the inferior AV cushion (iAVC) endothelium and mesenchyme, AV sulcus (arrow) and trabecular endothelium (arrowheads). Expression is absent in the myocytes (red). Nuclei—blue (Hoescht stain). LA-Left Atrium, RV-Right Ventricle, LV-Ventricle. Scale bars: A, B: 200 μm, C: 25 μm.

Figure 3. Filamin-A distribution in the E13.5 mouse heart.

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(A, D) H&E showing gross morphology. (B) Low magnification showing Filamin-A (green) expression in the atrioventricular septal complex (AVSC), the right and left lateral cushions (arrow heads), the AV sulcus (arrow), and epicardium (epi). (C) Higher magnification of boxed area in B showing Filamin-A expression in the endothelium and mesenchyme of the left lateral cushion (llAVC), the AV sulcus (arrow), interstitial mesenchyme (arrow heads), and ventricular trabeculae (asterisks), as well as the endothelium and mesenchyme of the fused superior and inferior AV cushions (left half of panel C). (E) Expression of Filamin-A (green) in the ventral aspect of the heart showing robust expression in the epicardium, the developing conal septum (boxed area) and weak expression in the developing arterial wall. (F) Higher magnification of boxed area in E showing Filamin-A expression in the mesenchyme of the fused conal cushion with no expression evident in adjacent MF20-positive myocytes (red). Nuclei—blue (Hoescht stain). Ao-Aorta, LA-Left Atrium, RA-Right Atrium, RV-Right Ventricle, LV-Ventricle. Scale bars: A, B: 200 μm, C: 25 μm.

At E17.5, filamin-A is present in all valve leaflets. Within the forming valves, expression is most intense in the interstitial cells on the atrial aspects of the inlet (mitral and tricuspid) leaflets and ventricular aspects of the arterial leaflets (Figure 4D and 5B,C). Expression is also robust in the mesenchyme of the left and right AV sulcus of the leaflets (arrow heads in 4C,D), and in the endothelium of the ventricular trabeculae (arrow heads in Figure 4E). At E17.5, the walls of the aorta and pulmonary arteries strongly express Filamin-A (Figure 5B). During neonatal life, filamin-A continues to be expressed in all of the leaflets with concentrated expression on the atrial aspect of the mitral leaflets and ventricular aspects of the semilunar valves (Figure 6). The epicardium, coronary vasculature, aortic and pulmonic walls, annulus fibrosae, and interstitial mesenchymal-like cells within the myocardial wall exhibit high levels of filamin-A expression. Importantly, by 6 months of age the expression of filamin-A in the leaflets was non-uniform and variable with low expression on both the ventricular and atrial endothelium with even weaker expression evident in subendothelial fibroblasts of the mitral and aortic leaflets. Expression however, remained robust in the coronary vasculature, the annulus fibrosae, and the chordae tendineae (Figures 7 and 8). There was no detectable expression of filamin-A in the papillary muscle or cardiac myocytes.

Figure 4. Cardiac Expression of Filamin-A at E17.5.

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(A) H&E showing gross morphology of the heart. (B) Low magnification of Filamin-A (green)/MF20 (red) immunostained heart showing intense staining of Filamin-A in the mitral and tricuspid valves (MV and TV, respectively (arrowheads)), and epicardium. Small letters “c, d, and e” in panel B are depicted in higher magnification in lower panels. (C, E) Filamin-A is expressed in the left and right AV sulcus (lAV, RAV sulcus), the epicardium (epi), non-myocytes present within the myocardial interstitium (arrowheads in panel C), and the endothelium of ventricular trabeculae (arrowheads in E). (D) Filamin-A expression in the forming mitral leaflets being present in both the mural mitral leaflet (mMiL) and the aortic leaflet of the mitral valve (AoLMiV). In these valve leaflets, expression is most intense on the endothelial surface of the ventricular aspects of the valves, with significantly weaker expression in the subendothelial mesenchyme/fibroblasts. Expression is absent within MF20 positive myocytes. Nuclei—blue (Hoescht stain). RA-right atrium, LA-left atrium, RV-right ventricle, LV-left ventricle, IVS-interventricular septum. Scale bars: A, B: 200 μm, CE: 25 μm.

Figure 5. Outflow tract expression of Filamin-A at E17.5.

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(A) H&E showing gross morphology of the E17.5 heart. (B) Low magnification of Filamin-A (green)/MF20 (red) expression cells demonstrating robust Filamin-A expression in the developing aortic leaflets (AoL), the dorsal aorta (Ao), and pulmonary vein (Pu). (C) Higher magnification of the aortic leaflets represented in panel B showing a gradient of endothelial and subendothelial mesenchyme/fibroblasts expression with more intense staining on the endothelial surface. Also, expression of Filamin-A is more robust on the ventricular aspect of the leaflets (arrows) versus the arterial side (arrows). Note intense expression of Filamin-A in the aortic wall (Ao) and no detectable expression in MF20 positive myocytes. (D) Higher magnification of boxed area in panel B showing epicardial (epi) expression of Filamin-A. Nuclei—blue (Hoescht stain). RA-right atrium, LV-left ventricle. Scale bars: A, B: 200 μm, C, D: 25 μm.

Figure 6. Cardiac expression of Filamin-A during neonatal development.

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(A) H&E showing gross morphology of the 1 week old neonatal heart. (B) Low magnification of Filamin-A (green)/MF20 (red) expression cells demonstrating robust Filamin-A expression in the aortic and pulmonic walls with significantly lower expression in the mitral, tricuspid and aortic valves. Small letters “c, d, and e” in panel B are depicted in higher magnification in lower panels. (C) Higher magnification of the mural mitral leaflet and the aortic leaflet of the mitral valve (mMIL and AoLMiV, respectively) showing a gradation of expression from base to tip with higher levels of Filamin-A detected in the base and mid regions of the leaflet. Additionally, subendothelial fibroblasts on the atrial aspect of the valve leaflets exhibit the highest level of Filamin-A. At this stage, very weak expression of Filamin-A is detected in the ventricularis at the tips of the mitral leaflets. (D) Filamin-A is most intense at the base of the mural mitral leaflet, in the annulus fibrosae (arrow) and the epicardium (epi). (E) Interstitial fibroblasts and coronary vasculature exhibit high levels of Filamin-A expression (arrowheads) with no detectable expression in the MF20 positive myocytes. Nuclei—blue (Hoescht stain). RA-right atrium, LA-left atrium, RV-right ventricle, LV-left ventricle, IVS-interventricular septum. Scale bars: A,B: 200 μm, C–E: 25 μm.

Figure 7. Adult cardiac expression of Filamin-A.

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(A) H&E showing gross morphology of the 3 month-old adult heart. (B) Low magnification of Filamin-A (green)/MF20 (red) expression showing robust Filamin-A expression in the aortic wall (arrow) and weak expression in the Mitral and Aortic leaflets (AoL). (C) Higher magnification of the tips of the mitral leaflet showing overall weak expression throughout the leaflet. (D) Expression of Filamin-A is most intense in the endothelial cells at the base of the mitral leaflet, and in fibroblasts intercalated at the annulus/myocardial border (arrowheads). Weak expression is evident in the annulus fibrosae. (E) Expression in the myocardial wall is restricted to the coronary vasculature (arrowheads). Nuclei—blue (Hoescht stain). LV-left ventricle, IVS-interventricular septum, AoLMiV-Aortic leaflet of the mitral valve, mMiL-Mural Mitral Leaflet. Scale bars: A,B: 200 μm, C–E: 25 μm.

Figure 8. Whole mount confocal analysis of Filamin-A in adult mural mitral leaflet.

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(A) Microdissected entire mitral leaflet showing septal, mural, and mitroaortic regions. Valve leaflet, chordae tendineae (ct), annulus fibrosa, and papillary muscles (PM) are denoted. (B) Whole mount immunolocalization of boxed area in panel A showing nonuniform expression of Filamin-A in the ventricularis endothelium of the adult mural mitral leaflet (green). Endothelial cells and fibroblasts within the chordae tendineae exhibit uniform distribution of Filamin-A expression. No expression of Filamin-A is evident within the papillary muscle. Asterisks represent autofluorescent blood cells. (C) Higher magnification of the chordae tendineae represented in panel B. Scale bars: A: 500μm, B: 100μm, C: 40μm.

Filamin-A expression was investigated in a myxomatous mitral leaflet isolated from a patient who had undergone valve replacement surgery and compared to that of a non-diseased normal human mitral valve (Figure 9). Movats pentachrome stain demonstrates clear evidence of a myxoid degeneration with defects in stratification of the fibrosa, spongiosa, and atrialis layers (Figure 9A) as compared to the normal mitral valve (Figure 9E). Immunohistochemical analysis for α-SMA and Filamin-A expression indicate extensive activation of these two genes throughout the entire valve leaflet, whereas the control leaflet demonstrates limited expression in the mid-portion of the leaflet being restricted to subendothelial valve fibroblasts of the atrialis (compare Figure 9B–D with F–H).

Figure 9. Movat’s Pentachrome and Immunohistochemical Analysis of Myxomatous and Normal Human Mitral Valve Leaflets.

Figure 9

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(A, E) Movat’s pentachrome staining of human myxomatous and normal mitral valve leaflets with collagen (yellow), elastin (black/orange), and proteoglycans (blue-green). Notice the myxomatous valve exhibits significant disorganization of the normal trilaminar structure with disrupted/diminished elastin, excess proteoglycan accumulation, and randomized collagen distribution (A-atrialis, V-ventricularis). (B–D, F–H) Immunohistochemical localization of α-SMA (green) and Filamin-A (red) demonstrating excessive production of these two proteins throughout the myxomatous valve whereas the control valve exhibits co-expression (yellow) in the mid-portion of the leaflet being restricted to subendothelial valve fibroblasts of the atrialis. Hoescht-nuclei (blue)

Discussion and Conclusions

Mutations in the Filamin-A gene were recently shown by genetic studies to cause an X-linked form of myxomatous valvular dystrophy in humans (Kyndt et al.; 2007; Kyndt et al.; 1998). Although much is currently known about this cytoskeletal protein, relatively nothing is known about its role(s) in cardiac morphogenesis. This is likely due, in part, to the lack of a thorough study of the tempero-spatial pattern of filamin-A during valve morphogenesis. An understanding of this expression pattern will provide an important step in understanding how filamin-A functions during cardiac development, and may provide insight into how mutations in this gene can result in myxomatous valvular dystrophy. Thus, we performed an immunohistochemical analysis of filamin-A protein expression throughout murine embryonic and postnatal cardiac development. During embryonic development, filamin-A is found in the endothelium and mesenchyme of AV and OFT cushions, the epicardium, the AV sulcus, migrating epicardial-derived cells (EPDCs), the endothelium of the ventricular trabeculae, and neural crest cells. Expression is, however, never detected in the myocardium by either immunohistochemistry or in situ hybridization (data not shown). Cell tracing experiments using mouse models that will detect neural crest derived cells (Wnt-1 Cre/ROSA26), endothelium-derived cells (Tie2-Cre/ROSA26) and epicardium and epicardial-derived cells (WT1-Cre/ROSA26) demonstrated that Filamin-A is expressed by each of these valve precursor populations (Supplemental Figures 1–3). However, the role of Filamin-A in these valve progenitor cells is unknown.

Functional analyses through gene knockout studies have provided important, yet limited information, as to the role of filamin-A during cardiac morphogenesis (Feng et al.; 2006; Hart et al.; 2006). Hemizygous mice for the filamin-A null allele show embryonic lethality and a wide range of cardiovascular malformations, including: incomplete septation of the outflow tract leading to a common arterial trunk (persistent truncus arteriosis), abnormally thickened and malformed OFT valves, atrial and ventricular septal defects, type B interruption of the aortic arch, abnormal endothelial organization in blood vessels, abnormal vascular permeability, and thickening of the mitral valve. Compensation by the other filamin genes does not seem to occur, nor have cardiovascular defects been described in mouse mutants for either filamin-B or filamin-C. The pleiomorphic array of cardiac malformations and the expression of filamin-A during development would seemingly support filamin-A as having an effect on a multitude of different cell types including neural crest, cushion/valve endocardium, cushion/valve mesenchyme/fibroblasts, and epicardium, which are supported by our data.

In the endothelium of the developing valves and vasculature, it has been proposed that filamin-A is important for maintaining and stabilizing cell-cell contacts and adherens junctions. Lack of filamin-A protein causes loss or disorganization of PECAM and VE-Cadherin resulting in cell-cell instability and endothelial sloughing. Ultrastructural analyses confirmed abnormal endocardial adherens junctions and are a proposed cause of embryonic lethality in the filamin-A mutant mice (Feng et al.; 2006; Hart et al.; 2006). Whether this putative role of filamin-A is specific for the endocardial epithelium, or can be extrapolated to all epithelia, such as the epicardial epithelium, in which filamin-A is intensely expressed, is unknown.

The defects observed in the outflow tract (i.e. persistent truncus arteriosis, interruption of the aortic arch type B) implicate a significant role for filamin-A in regulating neural crest behavior. However, when filamin-A was removed from neural crest cells using the neural crest-specific Wnt-1 Cre/ROSA26 mouse line, neural crest cell migration appeared normal. Thus, it was proposed that normal neural crest “function” was disrupted once the filamin-A deficient cells reach the OFT cushions of the heart (Feng et al.; 2006). Interestingly, as we demonstrate, filamin-A expression in the extracardiac neural crest cells is relatively weak. Once these cells have migrated into the OFT, filamin-A expression intensified, suggestive of a more crucial role for filamin-A-mediated neural crest cell behavior within the outlet cushions.

The neural crest cells, cushion endothelium, and epicardial epithelium all express high levels of filamin-A and each of these cell types undergo an endothelial-mesenchymal transformations (EMT) (Dettman et al.; 1998; Gittenberger-de Groot et al.; 1998; Perez-Pomares et al.; 1997; Wessels and Perez-Pomares; 2004). However, data from the filamin-A knock-out mice demonstrate that mesenchyme are present within the AV and OFT cushions, precluding a major role for filamin-A in regulating the EMT process in the cushions. Our data suggest that a major role for filamin-A in the developing valves is two fold: (i) to maintain endothelial integrity and (ii) promote maturation of the valvular interstitium after EMT (post-EMT). This is further substantiated by filamin-A knockout mice, which die during post-EMT valve maturation at E14.5 (Feng et al.; 2006) or E15.5 (Hart et al., 2006). Importantly, filamin-A expression significantly diminishes in the valve leaflets during postnatal life, suggesting a significant role for filamin-A in regulating embryonic valve maturation during a defined window of post-EMT valve remodeling. The presence of abnormally thickened and malformed OFT valves at E14.5 (Feng et al.; 2006) and dysplastic mitral valves at E15.5 (Hart et al., 2006) in the filamin-A null mice are consistent with this hypothesis.

To date Filamin-A is the only gene demonstrated as harboring causal mutations in patients with non-syndromous myxomatous valvular dystrophy. One major question to pose is why these specific point mutations found in the amino region of the protein cause only valve specific alterations in humans? Interestingly, mutations in other regions of the Filamin-A protein cause a wide spectrum of congenital anomalies including: Melnick-Needles syndrome, frontomeatphyseal dysplasia, otopalatodigital syndrome, and periventricular heterotopia (Zhou et al.; 2010). To date, the molecular mechanisms by which Filamin-A functions during valve morphogenesis are currently unknown, and more importantly how the mutations in Filamin-A that result in X-linked myxoid valvular dystrophy (XMVD) function to cause the disease, are also unknown. However, clues on how these mutations (and normal filamin-A) function during valve development may come from previous reports that filamin proteins appear to serve as hubs, or docking platforms, for second messengers important in signal transduction. To date, over 70 proteins have been identified as being interacting partners with filamin proteins (Zhou et al.; 2010). Some of these filamin-A-interacting proteins have been demonstrated as playing a crucial role in promoting TGFβ and/or BMP signals. These interactions promote phosphorylation and nuclear accumulation of downstream TGFβ and BMP mediators such as Smads2, 3, and 5 (Sasaki et al.; 2001). By regulating these growth factor-mediated responses during valve development, filamin-A could play an essential role in regulating how the valve is built. Clinically, it remains to be determined whether the Filamin-A point mutations found in patients with XMVD positively or negatively affect growth factor signaling. However, it is interesting to note that Fibrillin-1 mutations, which cause Marfan syndrome, result in disrupted TGFβ signaling in adult valves, contributing to myxomatous valves and mitral valve prolapse (Neptune et al.; 2003; Ng et al.; 2004; Robinson et al.; 2006). In addition to transmitting growth factor regulated signals, filamin-A has been shown to be a necessary regulator of collagen compaction by instilling tension on collagen fibrils, resulting in active remodeling of the surrounding matrix (Gehler et al.; 2009). This regulation is mediated, in part, by the interaction between filamin-A and integrin β1 (Kim et al.; Kim et al.; 2008), which together promote assembly of a mechanosensitive complex, that bidirectionally senses the tension of the matrix and regulates cellular contractility during embryonic morphogenesis. Through these interactions, filamin proteins can produce enough traction forces to remodel their surrounding extracellular environment, implicating this class of proteins as “mechanotranducers” (Gehler et al.; 2009; Glogauer et al.; 1998; Jiang and Campbell; 2008; Meyer et al.; 1998; Pentikainen and Ylanne; 2009). During post-EMT valve maturation, remodeling of the surrounding valvular microenvironment via collagen compaction is a required biophysical response necessary to achieve a mature valvar form (Butcher et al.; 2007). Because collagen compaction is imparted via actin-cytoskeletal remodeling (Farsi and Aubin; 1984), actin and filamin-A may play a significant role in valve remodeling events due to their ability to promote collagen compaction, although data specifically showing this has not yet been demonstrated.

In addition to regulating collagen compaction during valve maturation, recent work implicates filamin-A and its close association with cortical actin in regulating collagen deposition. This work, initially performed in the tendon, resulted in the discovery of a cellular structure termed “fibripositor” (Canty et al.; 2004; Canty et al.; 2006; Humphries et al.; 2008; Kapacee et al.; 2008). Fibripositors are defined as actin-stabilized plasma membrane processes that protrude into the ECM to deposit and organize newly assembled collagen fibrils in parallel alignment with the existing matrix (Canty et al.; 2006). Importantly, each of the Filamin-A mutations identified to date that result in non-syndromous MVP are clustered near the actin-binding domain, and are anticipated to disrupt actin stabilization (Kyndt et al.; 2007). This disruption could ultimately compromise fibripositor formation resulting in aberrant collagen production, deposition and faulty organization of the developing valve.

The potential for Filamin-A mutations in humans to disrupt these two processes (matrix compaction and collagen deposition), would be anticipated has having profound effects on the biomechanical function of the cardiac valves. Reports by Grande-Allen et al. (2003) (Grande-Allen et al.; 2003) have demonstrated that myxomatous mitral valves and chordae tendineae are biomechanically weaker and more compromised than unaffected valves. This is largely due to biochemical changes in glycosaminoglycans, elastin integrity, and collagen production/distribution. These biochemical changes likely stimulate secondary pathways that lead to further degeneration of the mitral valves and their suspensory apparatus. Coincident with these changes are the observation of α-SMA positive “activated myofibroblasts” in myxomatous valves (Rabkin et al.; 2001; Rabkin- Aikawa et al.; 2004). Of relevance, we provide evidence that Filamin-A expression is upregulated in human myxomatous mitral valve leaflets and partially co-localizes with α-SMA positive myofibroblasts. It is interesting to note that not all Filamin-A expressing cells are α-SMA positive indicating presence of Filamin-A in valve disease may reflect a larger population of altered cell phenotype. Additionally, α-SMA (Hinton et al.; 2006) and filamin-A (this report) are expressed during normal valve development, and are significantly down-regulated in the mature mitral valve. However, in the context of human myxomatous valves, we demonstrate that these genes are highly up-regulated, and thus consistent with the concept that mitral valve disease may reactivate a developmental process.

We hypothesize that filamin-A mutations, present at conception, result in defects in the molecular machinery essential for TGFβ-mediated signaling, collagen deposition and matrix remodeling; ultimately affecting the biomechanical stability of the developing mitral valve. The generation and analysis of unique point mutation knock-in mouse models, in addition to the selective removal of filamin-A in valve progenitor cells (endothelium, neural crest, and/or epicardium), will ultimately test these hypotheses and provide mechanistic insight into pathways governing both normal post-EMT valve development and valve pathogenesis.

Supplementary Material

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Supplementary Data

Acknowledgments

NIH-NHLBI: HL33756 (RRM), NIH-NCRR: COBRE P20RR016434-07 (RRM and BSH), P20RR016434-09S1 (RRM and RAN); National Science Foundation: FIBRE EF0526854 and EPS-0902795 (RRM and RAN); the Foundation Leducq (Paris, France) Transatlantic Mitral Network of Excellence grant 07CVD04 (RAN, RRM, RAL, AH, SAS, JM, JJS); SC INBRE: 5MO1RR001070-28 (RAN); INSERM, UMR915, Nantes, F-44035, France (JM and JJS); CNRS, ERL3147, Nantes F-44035, France (JM and JJS); Université de Nantes, Faculté de Médecine, l’institut du thorax, Nantes, F-44035, France (JM and JJS); CHU Nantes, l’institut du thorax, Nantes, F-44000, France (JM and JJS). NIHNCRR-P20 RR016434, NIH-HLBI 1R01-HL084285, AHA-GIA 09GRNT2060075(AW), AHA-GIA 0835460N (EA)

The authors would like to thank members of the Markwald and Norris labs for critical reading of the manuscript, and Aimee Phelps for excellent technical assistance.

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