Active adaptation of the tethered mitral valve: insights into a compensatory mechanism for functional mitral regurgitation

Abstract

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 61+/-6 days compared with 6 unstretched sheep MVs. Total diastolic leaflet area increased by 2.4+/-1.3 cm(2) (17+/-10%) from 14.3+/-1.9 to 16.7+/-1.9 cm(2) (P=0.006) with stretch with no change in the unstretched valves despite sham open heart surgery. Stretched MVs were 2.8 times thicker than normal (1.18+/-0.14 versus 0.42+/-0.14 mm; P<0.0001) at 60 days with an increased spongiosa layer. Endothelial cells (CD31(+)) coexpressing alpha-smooth muscle actin were significantly more common by fluorescent cell sorting in tethered versus normal leaflets (41+/-19% versus 9+/-5%; P=0.02), indicating endothelial-mesenchymal transdifferentiation. alpha-Smooth muscle actin-positive cells appeared in the atrial endothelium, penetrating into the interstitium, with increased collagen deposition. Thickened chordae showed endothelial and subendothelial alpha-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.

Figure 1

Normal MV closure (A,C); effect of apical and posterior shift of PMs restraining the MV, causing MV leaflet tethering (D) and, if severe enough, MR (B). Lower panel (C & D): Echocardiography of the LV (apical two-chamber view) in the same sheep prior to (C) and after pulling the PMs apically resulting in MV leaflet tethering (D). The dashed line indicates the mitral annular level. (Ao: Aorta, LA: left atrium; LV: left ventricle, MR: mitral regurgitation; MV: mitral valve; PM: papillary muscle) (Figures A & B adapted from Levine et al 14).

Figure 2

Normal MV (upper panel) versus stretched MV leaflets (lower panel), which are less opaque consistent with increased thickness. (PM: papillary muscle; units in cm).

Figure 3

Hematoxylin & eosin (HE) and Masson staining in the normal (left) and stretched MV (right) demonstrating increased spongiosa layer thickness. (Blue=collagen)

Figure 4

A&B: Stretched chordae are significantly thicker and have decreased collagen alignment and density compared to unstretched chordae. C&D: Stretched chordae show endothelial-cell α-SMA-positive staining and subendothelial accumulation of α-SMA-positive myofibroblasts.

Figure 5

Increased endothelial cells (CD31+) also expressing α-SMA in stretched versus unstretched MVs (A,B). Left: representative sample of cells double-labeled with isotype-matched control antibodies. Right: cells double-labeled with anti-sheep CD31 conjugated to FITC and anti-α-SMA conjugated to phycoerthrin (PE). Compensation was performed with singly labeled cells (not shown). C&D: MV ECs undergo TGF-β-induced EMT in vitro. C: Immunostained MV ECs showing CD31 localized at cell-cell borders and α-SMA was undetectable (inset), confirming endothelial phenotype. Scale bar, 50μm. D: Western blots of lysates from MV ECs treated with 1ng/ml TGF-β1, TGF-β2, or TGF-β3 for 5 days. All three TGF-β isoforms induced expression of α-SMA.

Figure 6

Left: Unstretched MV showing negative α-SMA staining along the CD31+ endothelium. Right: α-SMA+ staining in the atrial endothelium (also CD31+) of a stretched MV, with nests of α-SMA+ cells appearing to penetrate the interstitium (asterisks, below). Lower panel: Schematic of active mitral valve adaptation by EMT (adapted from Armstrong et al 36). (α-SMA: α-smooth muscle actin)

Figure 7

Stretched MV: α-SMA-positive cells penetrating from the atrial surface into the valve interstitium (A) with increased collagen deposition in the same region (B).

Figure 8

α-SMA-positive cells in a patient with ischemic MR in the atrial endothelium (left, arrows) and throughout the interstitium (asterisks).