Spatial and Temporal Analysis of Extracellular Matrix Proteins in the Developing Murine Heart: A Blueprint for Regeneration

Abstract

The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.

Three-dimensional scaffolds of fetal decellularized hearts exhibit enhanced potential to support cardiac cells in comparison to the adult

“…Similar to others reports [20,50], our findings show that the fetal myocardium, when compared to the adult, is abundant in fibronectin and rather scarce in collagen type I. The preponderance of fibronectin in fetal tissues is likely the best illustration of the high instructive microenvironment established in heart development, because this molecule orchestrates multiple fundamental cellular processes such as adhesion, migration, proliferation and differentiation [43,51].…”

“…These observations are consistent with the maturation of basement membrane that occurs in late developmental stages [28][29][30]. Interestingly, the pattern of laminin and collagen type IV of decellularized heart matrices resembled their distribution in the native tissue ( Fig.…”

Fibrosis-Related Gene Expression in Single Ventricle Heart Disease

“…Given known developmental upregulation and accumulation of collagen during the neonatal period 17, 18 , infant patients with single ventricle. were analyzed separately from the Stage III pediatric subjects, which also served to minimize changes that may arise solely from differences in RV volume load.…”

Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells

“…[37][38][39][40][41] In contrast, Eln endogenous fluorescence has not been routinely used and the small number of existing studies utilize a variety of excitation and emission imaging parameters, with limited validation of specificity for Eln protein. 37,[42][43][44][45][46][47] Because of the discrepancies among optimal imaging parameters for Eln, it was essential to compare the endogenous Eln signal with immunofluorescence using an anti-Eln antibody with MPLSM.…”