Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro

Abstract

Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

Figure 1

Cell strain. CPCs were seeded on the appropriate matrix for 6 hours. Video microscopy (19 frames per second) captured the motion of beads tethered to CPCs under cyclic 1 Hz strain. (a) Representative distance tracings from CPCs cultured on cECM; programmed strain is reported on graph. (b) Strain magnitude effects on measured strain. One-way ANOVA with Tukey's multiple comparison test; bars represent mean + SEM; ^# p < 0.05, ^## p < 0.01, and ^#### p < 0.001; n = 3–10; 3.5, 7.0: strain magnitude (%), PLL (brown): poly-L-lysine, LN (green): laminin, FN (purple): fibronectin, COL (blue): collagen I, and cECM (red): naturally derived cardiac extracellular matrix.

Figure 2

Strain improves VEGF secretion in CPC conditioned media. CPCs were seeded on each matrix for 6 hours and then cyclic strain was applied for 24 hours. Conditioned media were evaluated by ELISA. One-way ANOVA with Tukey's multiple comparison test; bars represent mean + SEM; ^∗ p < 0.05; n = 3-4; x-axis: 0–15: strain magnitude (%) and VEGF: vascular endothelial growth factor A.

Figure 3

Fibronectin increases CPC division at low strain magnitudes. CPCs were seeded on each matrix for 6 hours and then cyclic strain was applied for 24 hours. (a) Representative image showing nuclear condensation and cytokinesis. Blue: DAPI and green: FITC-maleimide. Insert shows magnification of a dividing cell. (b) Representative image of aurora B kinase staining. Blue: DAPI, green: FITC-maleimide, and red: aurora B kinase. (c) Quantified results; one-way ANOVA with Tukey's multiple comparison test; bars represent mean + SEM; ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001; n = 4–6; PLL (brown): poly-L-lysine, LN (green): laminin, FN (purple): fibronectin, COL (blue): collagen I, and cECM (red): naturally derived cardiac extracellular matrix.

Figure 4

FN induces PCNA expression. CPCs were seeded on each matrix for 6 hours followed by 24 hours of cyclic strain and lysed. Cell lysate was evaluated by western blot. (a) Representative blots. (b) Quantification by densitometry; n = 4-5; PLL (brown): poly-L-lysine, LN (green): laminin, FN (purple): fibronectin, COL (blue): collagen I, cECM (red): naturally derived cardiac extracellular matrix, PCNA: proliferating cell nuclear antigen, and ∗ represents p < 0.05 by ANOVA.

Figure 5

CPC connexin43 expression is highest at low strain magnitudes. CPCs were seeded on each matrix for 6 hours and then cyclic strain was applied for 24 hours. Connexin43 expression was assessed by western blot. (a) Representative blots and (b-c) densitometry quantification. One-way ANOVA with Tukey's multiple comparison test; bars represent mean + SEM; ^∗ p < 0.05; n = 4–7. (b) Matrix-dependent effects. (c) Strain magnitude-dependent effects. PLL (brown): poly-L-lysine, LN (green): laminin, FN (purple): fibronectin, COL (blue): collagen I, cECM (red): naturally derived cardiac extracellular matrix, 0–15: strain magnitude (%), and Cnx43: connexin43.

Figure 6

Strain induces CPC alignment. CPCs were seeded on each matrix for 6 hours and then cyclic strain was applied for 24 hours. (a) Quantification of strain-induced matrix-dependent alignment. Kruskal-Wallis one-way ANOVA with Dunn's multiple comparison tests; bars represent mean + SEM; ^∗ p < 0.05 and ^∗∗ p < 0.01; n = 4–7; PLL (brown): poly-L-lysine, LN (green): laminin, FN (purple): fibronectin, COL (blue): collagen I, and cECM (red): naturally derived cardiac extracellular matrix. (b) Representative cell strain images. Blue: DAPI and green: FITC-maleimide; arrow indicates principle direction of strain; 0–15: strain magnitude (%).