Background-In patients with left ventricular infarction or dilatation, leaflet tethering by displaced papillary muscles frequently induces mitral regurgitation, which doubles mortality. Little is known about the biological potential of the mitral valve (MV) to compensate for ventricular remodeling. We tested the hypothesis that MV leaflet surface area increases over time with mechanical stretch created by papillary muscle displacement through cell activation, not passive stretching. Methods and Results-Under cardiopulmonary bypass, the papillary muscle tips in 6 adult sheep were retracted apically short of producing mitral regurgitation to replicate tethering without confounding myocardial infarction or turbulence. Diastolic leaflet area was quantified by 3-dimensional echocardiography over 61Ϯ6 days compared with 6 unstretched sheep MVs. Total diastolic leaflet area increased by 2.4Ϯ1.3 cm 2 (17Ϯ10%) from 14.3Ϯ1.9 to 16.7Ϯ1.9 cm 2 (Pϭ0.006) with stretch with no change in the unstretched valves despite sham open heart surgery. Stretched MVs were 2.8 times thicker than normal (1.18Ϯ0.14 versus 0.42Ϯ0.14 mm; PϽ0.0001) at 60 days with an increased spongiosa layer. Endothelial cells (CD31 ϩ ) coexpressing ␣-smooth muscle actin were significantly more common by fluorescent cell sorting in tethered versus normal leaflets (41Ϯ19% versus 9Ϯ5%; Pϭ0.02), indicating endothelial-mesenchymal transdifferentiation. ␣-Smooth muscle actin-positive cells appeared in the atrial endothelium, penetrating into the interstitium, with increased collagen deposition. Thickened chordae showed endothelial and subendothelial ␣-smooth muscle actin. Endothelial-mesenchymal transdifferentiation capacity also was demonstrated in cultured MV endothelial cells. Conclusions-Mechanical stresses imposed by papillary muscle tethering increase MV leaflet area and thickness, with cellular changes suggesting reactivated embryonic developmental pathways. Understanding such actively adaptive mechanisms can potentially provide therapeutic opportunities to augment MV area and reduce ischemic mitral regurgitation. (Circulation. 2009;120:334-342.)Key Words: echocardiography Ⅲ mitral valve Ⅲ valves I n population studies, valvular heart disease is common, with mitral regurgitation (MR) most prevalent. 1 Although degeneration is the leading cause of MR surgical repair, 2 coronary artery disease with myocardial infarction and left ventricular (LV) dysfunction frequently causes functional MR as a result of global LV remodeling and sphericity 3-5 or localized inferoposterior wall remodeling 6 -12 ; both cause apical, posterior, and outward displacement of the papillary muscles (PMs) 6 -12 and mitral valve (MV) leaflet tethering 13 that prevents effective closure ( Figure 1A and 1B). 12,14 Editorial see p 275 Clinical Perspective on p 342Patients who develop MR after myocardial infarction or with congestive heart failure, even after surgical or catheter revascularization, have doubled mortality and in- To observe leaflet adaptation to tethering over time, we asse...

BACKGROUND In patients with myocardial infarction (MI), leaflet tethering by displaced papillary muscles induces mitral regurgitation (MR), which doubles mortality. Mitral valves (MVs) are larger in such patients but fibrosis sets in counterproductively. The investigators previously reported that experimental tethering alone increases mitral valve area in association with endothelial-to-mesenchymal transition. OBJECTIVES This study explored the clinically relevant situation of tethering and MI, testing the hypothesis that ischemic milieu modifies MV adaptation. METHODS Twenty-three adult sheep were examined. Under cardiopulmonary bypass, the PM tips in 6 sheep were retracted apically to replicate tethering, short of producing MR (tethered-alone). PM retraction was combined with apical MI created by coronary ligation in another 6 sheep (tethered + MI), and left ventricular (LV) remodeling was limited by external constraint in 5 additional sheep (LV constraint). Six sham-operated sheep were controls. Diastolic MV surface area was quantified by 3-dimensional echocardiography at baseline and after 58 ± 5 days, followed by histopathology and flow cytometry of excised leaflets. RESULTS Tethered + MI leaflets were markedly thicker than tethered-alone valves and sham controls. Leaflet area also increased significantly. EMT, detected as α-smooth muscle actin-positive endothelial cells, significantly exceeded that in tethered-alone and control valves. Transforming growth factor-β, matrix metalloproteinase expression, and cellular proliferation were markedly increased. Uniquely, tethering + MI showed endothelial activation with vascular adhesion molecule expression, neovascularization, and cells positive for CD45, considered a hematopoietic cell marker. Tethered + MI findings were comparable with external ventricular constraint. CONCLUSIONS MI altered leaflet adaptation, including a profibrotic increase in valvular cell activation, CD45-positive cells, and matrix turnover. Understanding cellular and molecular mechanisms underlying leaflet adaptation and fibrosis could yield new therapeutic opportunities for reducing ischemic MR.

Endothelial-mesenchymal transformation (EMT) is a critical event for the embryonic morphogenesis of cardiac valves. Inducers of EMT during valvulogenesis include VEGF, TGF-β1, and wnt/β-catenin (where wnt refers to the wingless-type mammary tumor virus integration site family of proteins), that are regulated in a spatiotemporal manner. EMT has also been observed in diseased, strain-overloaded valve leaflets, suggesting a regulatory role for mechanical strain. Although the preponderance of studies have focused on the role of soluble mitogens, we asked if the valve tissue microenvironment contributed to EMT. To recapitulate these microenvironments in a controlled, in vitro environment, we engineered 2D valve endothelium from sheep valve endothelial cells, using microcontact printing to mimic the regions of isotropy and anisotropy of the leaflet, and applied cyclic mechanical strain in an attempt to induce EMT. We measured EMT in response to both low (10%) and high strain (20%), where low-strain EMT occurred via increased TGF-β1 signaling and high strain via increased wnt/β-catenin signaling, suggesting dual strain-dependent routes to distinguish EMT in healthy versus diseased valve tissue. The effect was also directionally dependent, where cyclic strain applied orthogonal to axis of the engineered valve endothelium alignment resulted in severe disruption of cell microarchitecture and greater EMT. Once transformed, these tissues exhibited increased contractility in the presence of endothelin-1 and larger basal mechanical tone in a unique assay developed to measure the contractile tone of the engineered valve tissues. This finding is important, because it implies that the functional properties of the valve are sensitive to EMT. Our results suggest that cyclic mechanical strain regulates EMT in a strain magnitude and directionally dependent manner.tight junctions | cytokines | activated myofibroblast C ardiac valves are sophisticated structures that function in a complex mechanical environment, opening and closing more than 3 billion times during the average human lifetime (1). Initially considered passive flaps of tissue, it is now acknowledged that valves contain a highly heterogeneous population of endothelial (VEC) and interstitial (VIC) cells. The VICs exist as synthetic, myofibroblast, or smooth muscle-like phenotypes (2, 3) and alter their tone in response to vasoactive mediators (4-7). The VECs line the surface of the valve leaflet and are unique in their ability to undergo endothelial-mesenchymal transformation (EMT), a process that is crucial for valvulogenesis (8, 9). Recent clinical evidence of EMT has been observed in pathologies such as ischemic cardiomyopathy and concomitant mitral regurgitation and is correlated with increased leaflet mechanical strains (10, 11). These pathological strains can be oriented obliquely to cell and tissue orientation (12, 13), suggesting the possible interaction between mechanical forces and tissue architecture in regulating EMT.Prior work has focused on the regulation of ...