Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro

Abstract

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development, and studies suggest that it plays an important role in regulating myocyte proliferation. Here, the effects of fetal, neonatal and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro are studied. At 24h, overall cell attachment was lowest on fetal ECM; however, ~80% of the cells were cardiomyocytes, while many non-myocytes attached to older ECM and poly-l-lysine controls. After 5 days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared with other substrates at 5 days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.

Keywords: Cardiac tissue engineering; Cardiomyocyte; Extracellular matrix; Proliferation; Second Harmonic Generation imaging.

Figure 1. Changes in cardiac cell proliferation and ECM composition with developmental age

(A) Example images of fetal, neonatal, and adult heart tissue sections at 20× magnification. Tissue sections are stained for nuclei with Hoechst (blue), the mitosis marker phospho-histone H3 (green), and cardiac α-actin (red). (B) Total cell proliferation drops significantly in the adult heart. (C) Cardiomyocyte-specific proliferation is highest in the fetal heart. *indicates significant difference for p < 0.05. Scale bars = 100 μm. (D) ECM composition is represented as the percentage of the 15 most abundant proteins detected by LC-MS/MS at each developmental age.

Figure 2. Decellularization of cardiac ECM

(A) Native hearts are shown from fetal pups at E18, neonatal pups at P2, and an adult rat. Fetal and neonatal hearts are shown with a dime while the adult heart is shown with fetal hearts for size comparison. (B) Hearts after decellularization with SDS and TritonX-100 wash. Tissue has turned white, indicating removal of cells. (C) Second harmonic generation image volume projections of decellularized hearts. Fetal ECM has smaller fibers and lower average signal compared to neonatal and adult hearts. Scale bar = 50 μm.

Figure 3. Optimization of fetal heart decellularization

(A) Representative images of fetal hearts after decellularization with SDS or TritonX-100 at different concentrations. (B) DNA assay shows significant decrease in cellular content after decellularization. ELISA results for (C) Collagen I, (D) Collagen V, and (E) Fibronectin show differences in ECM preservation with different decellularization methods. *indicates significant difference for p < 0.05.

Figure 4. Cell adhesion on cardiac ECM at 24 hr

(A) Representative images of cardiomyocytes stained for sarcomeric α-actinin on PLL and ECM-coated substrates. (B) Cell density (blue) was highest on PLL substrates and lowest on fetal ECM. Cardiomyocyte density (brown) was highest for FBS conditions, but the majority of cells were myocytes on fetal ECM. (C) The cardiomyocyte population as a percentage of all cells shows that specific adhesion was greatest on fetal ECM and lowest on PLL serum free. $ = significant difference vs. all ECM. # = significant difference vs. all other conditions.

Figure 5. Cell response on cardiac ECM at 5 days

(A) Representative images of ventricular on PLL and ECM-coated substrates. Nuclei stained with Hoechst (blue) and cells of the myocyte lineage stained with cardiac α-actin (red). (B) Total cell density (blue) is highest on PLL with FBS stimulation. Cardiomyocyte density (brown) was significantly higher on fetal and neonatal ECM compared to FBS, PLL, and adult ECM. (C) The cardiomyocyte population as a percentage of all cells shows that fetal ECM maintained high purity of myocytes over time in culture. # = significant difference vs. all other conditions. $ = significant difference vs. fetal and neonatal ECM.

Figure 6. Proliferation on ECM

(A) Fold change in total cell numbers (blue) and cardiomyocytes (brown) from 24 hr to 5 days. Cardiomyocyte numbers had greatest increase on fetal ECM compared to all other conditions, followed by neonatal ECM. (B) Staining for PHH3 shows no significant difference in the overall proliferating cell population (blue); however, significantly more myocytes (brown) were PHH3+ on fetal ECM compared to other conditions. (C) Representative images of cells stained for PHH3 (green) and cardiac α-actin (red) on PLL and ECM (arrows indicate PHH3+ nuclei). # = significant difference vs all other conditions. $ = significant difference vs FBS stimulated and serum-free PLL controls.

Figure 7. Consistent effects of ECM on cardiomyocytes

Data from a repeat experiment with different isolations of ECM and cells. (A) At 5 days, cardiomyocyte density is significantly higher on fetal ECM compared to PLL serum free and adult ECM. (B) Cardiomyocytes made up a greater percentage of the cell population on fetal ECM compared to FBS, PLL, and adult ECM. (C) Fold changes in cardiomyocyte number from 24 hr to 5 days were higher on fetal ECM compared to PLL serum-free and adult ECM. (D) Cardiomyocytes were a significantly greater portion of the proliferating cell population on fetal ECM compared to FBS and PLL. $ = significant difference vs FBS, PLL, and adult ECM.