The Three-Dimensional Microenvironment of the Mitral Valve: Insights into the Effects of Physiological Loads
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In the mitral valve (MV), numerous pathological factors, especially those resulting from changes in external loading, have been shown to affect MV structure and composition. Such changes are driven by the MV interstitial cell (MVIC) population via protein synthesis and enzymatic degradation of extracellular matrix (ECM) components.
While cell phenotype, ECM composition and regulation, and tissue level changes in MVIC shape under stress have been studied, a detailed understanding of the three-dimensional (3D) microstructural mechanisms are lacking. As a first step in addressing this challenge, we applied focused ion beam scanning electron microscopy (FIB-SEM) to reveal novel details of the MV microenvironment in 3D.
We demonstrated that collagen is organized into large fibers consisting of an average of 605 ± 113 fibrils, with a mean diameter of 61.2 ± 9.8 nm. In contrast, elastin was organized into two distinct structural subtypes: (1) sheet-like lamellar elastin, and (2) circumferentially oriented elastin struts, based on both the aspect ratio and transmural tilt. MVICs were observed to have a large cytoplasmic volume, as evidenced by the large mean surface area to volume ratio 3.68 ± 0.35, which increased under physiological loading conditions to 4.98 ± 1.17.
Our findings suggest that each MVIC mechanically interacted only with the nearest 3–4 collagen fibers. This key observation suggests that in developing multiscale MV models, each MVIC can be considered a mechanically integral part of the local fiber ensemble and is unlikely to be influenced by more distant structures.
KeywordsHeart valves Ultrastructure Valve interstitial cells Extracellular matrix Collagen Elastin
The authors would like to acknowledge Dr. Hua Gua (Rice University) and Dr. Dwight Romanovicz (UT Austin) for their assistance with the FIB-SEM and TEM instruments, as well as Sarah Poletti, Ethan Kwan, and Michelle Lu for their assistance with heart valve tissue isolation and preparation. This work was supported by the National Institutes of Health Grant [R01HL119297] to MSS and the American Heart Association Pre-Doctoral Fellowship [PRE33420135] to SA.
Conflict of interest
None of the authors of this work, Salma Ayoub, Karen C. Tsai, Amir H. Khalighi, and Michael S. Sacks, have a conflict of interest.
This article does not contain any studies with human participants or animal studies performed by any of the authors.
Video 1. Video of the 3D reconstruction of the mitral valve interstitial cell and its surrounding microenvironment showing the serial 2D FIB-SEM images (horizontal field width of 53.6 µm), followed by the 3D reconstruction of the nucleus, cytoplasm, collagen, and elastin. This video highlights the complexity of the interconnection between the valve interstitial cell and the surrounding extracellular matrix. Reconstruction of 77 serial sections (total thickness = 15.4 µm) with a slice thickness of 200 nm. Supplementary material 1 (MP4 102833 kb)
Video 2. Video of the 3D reconstruction of the mitral valve interstitial microenvironment showing the serial 2D FIB-SEM images (horizontal field width of 20.7 µm), followed by the 3D reconstruction of the collagen fibers and the elastin structures. Reconstruction of 30 serial sections (total thickness = 1.50 µm) with a slice thickness of 50 nm. Supplementary material 2 (MP4 94609 kb)
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