Epicardially secreted fibronectin drives cardiomyocyte maturation in 3D-engineered heart tissues

Abstract

Ischemic heart failure is due to irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could remuscularize infarcted hearts and improve cardiac function. However, these cardiomyocytes remained immature. Incorporating hPSC-derived epicardial cells has been shown to improve cardiomyocyte maturation, but the exact mechanisms are unknown. We posited epicardial fibronectin (FN1) as a mediator of epicardial-cardiomyocyte crosstalk and assessed its role in driving hPSC-derived cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). We found that the loss of FN1 with peptide inhibition F(pUR4), CRISPR-Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs resulted in immature cardiomyocytes with decreased contractile function, and inefficient Ca²⁺ handling. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Finally, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.

Keywords: cardiomyocytes; engineered heart tissues; epicardium; fibronectin; maturation.

Graphical abstract

Figure 1

Generation of 3D-EHTs with optimized cell ratios (A) Schematic for derivation of hESC-CMs. (B) Schematic for derivation of hESC epicardium. (C) Schematic for the generation of collagen-based 3D-EHTs containing hESC-CMs and EPIs and their subsequent histological and functional assessment. (D) Frank-Starling curves of active force generation of 3D-EHTs containing CM only, CM + 10% hESC epicardium, CM + 30% hESC epicardium, or CM+50% hESC epicardium. (E) Slope of active force generation. (F) Ca²⁺-upstroke velocity of 3D-EHTs containing CM only, CM + 10% hESC epicardium, CM + 30% hESC epicardium, or CM + 50% hESC epicardium. (G) Time to peak Ca²⁺-fluorescence in 3D-EHTs containing CM only, CM + 10% hESC epicardium, CM + 30% hESC epicardium, or CM + 50% hESC epicardium. (F and G) Presented calcium cycling data were taken from constructs paced at 1 Hz. Data presented as mean values; error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0001. Two-sided p values were calculated using a one-way ANOVA with post hoc correction for multiple comparisons. Each experimental group included N = 3 biological replicates.

Figure 2

Pharmacological inhibition of epicardial FN1 with pUR4 results in impaired FN1 binding to integrin α5β1 and reduced CM maturation (A) Schematic describing the mechanism of action of the recombinant peptide pUR4. PUR4 prevents FN1 from binding to the cell surface receptor integrin α5β1, hence preventing FN fibrillogenesis and signal transduction. (B) Marked reduction of FN1 expression at the protein level 72 h post treatment of hESC epicardium with pUR4. (C–E) 3D-EHTs containing CM alone or EPI + CM, untreated or treated with pUR4. (C) Confocal images showing FN1 deposition following treatment with pUR4. Scale bar, 5 μm. (D) Quantification of FN1 following inhibition with pUR4. (E) Confocal images showing expression of integrin α5β1 following treatment with pUR4. (F and G) Structural cardiac maturation as expressed by sarcomeric length (F) and sarcomere alignment (G) in 3D-EHTs containing CM only or CM + EPI with or without treatment with pUR4. (H and I) Frank-Starling curves of active force generation (H) and slopes of active force (I) of 3D-EHTs containing CM only, CM + pUR4, CM + EPI, or CM + EPI + pUR4. (J and K) Frank-Starling curves of passive force generation of 3D-EHTs containing CM only, CM + pUR4, CM + EPI, or CM + EPI + pUR4. (L–O) Ca²⁺ kinetics of 3D-EHTs containing CM only or CM + EPI with or without treatment with pUR4. All experimental groups had N = 3 biological replicates. (L and M) Ca2+ kinetics including upstroke velocity and time to peak fluorescence of 3D-EHTs containing CM only or CM+EPI with or without treatment with pUR4. (N and O) Ca2+ kinetics including time to 50% decay and time to 90% decay of 3D-EHTs containing CM only or CM+EPI with or without treatment with pUR4. Mean values; error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 3

Constitutive loss of epicardial FN1 using a Crispr-Cas9-edited KO hESC line results in impaired CM maturation (A) Cobblestone morphology and WT1 expression in Crispr-Cas9-edited KO FN1 epicardium. Scale bar, 20 μm. (B) RNA expression of the key epicardial markers WT1, TCF21, TBX18, and BNC1 in EPIs derived from H9-hESCs, RUES2-hESCs, and RUES2-hESC with KOFN. (C) Confocal images showing FN1 expression in day 10 epicardium in KOFN and WT EPIs respectively. Scale bar, 50 μm. (D) RNA expression of FN1 in H9-WT, H9-EPI, and RUES2-EPI. (E–Q) EPIs and CMs were derived from RUES2 hESC. (E and F) Confocal images showing FN1 (E) and α5β1 expression (F) in 3D-EHTs of constructs containing CM only, CM + EPI, or CM + KOFN EPI. Scale bar, 5 μm. (G) Quantification of FN1 expression by fluorescent intensity. (H and I) Structural cardiac maturation as expressed by sarcomeric length (H) and sarcomere alignment (I) in 3D-EHTs containing CM only, CM + EPI, or CM + KOFN EPI. (J and K) Frank-Starling curves of active force generation (J) and slopes of active force (K) of 3D-EHTs containing CM only, CM + EPI, or CM + KOFN EPI. (L and M) Frank-Starling curves of passive force generation (L) and slopes of passive force (M) of 3D-EHTs containing CM only, CM + EPI, or CM + KOFN EPI. (N–Q) Ca²⁺-kinetics of 3D-EHTs containing CM only, CM + EPI, or KOFN EPI. (E–Q) EPIs and CMs were derived from RUES2 hESC. All experimental groups had N = 4 biological replicates. Mean values; error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. Two-sided p values were calculated using a one-way ANOVA with post hoc correction for multiple comparisons.

Figure 4

Temporal loss of epicardial FN1 expression with a tetracycline-inducible KD is sufficient to abrogate CM maturation (A) RNA expression of the key epicardial markers WT1, TCF21, and BNC1 in three sOPTiKD-FN1 clones, with the highest expression in clone 5.3. (B) RNA expression in three sOPTiKD-FN1 clones following tetracycline exposure, with the greatest reduction in clone 5.3. (C) Cobblestone morphology and WT1 expression in clone 5.3 differentiated to D8 hESC epicardium following 24 h of tetracycline induction. Scale bar, 20 μm. (D) Confocal images showing FN1 expression in 3D-EHTs containing CM only, CM + sOPTiKD-FN1 EPI without tetracycline, and CM + sOPTiKD-FN1 EPI exposed to tetracycline. (E) Quantification of FN1 expression by fluorescent intensity. (F and G) Structural cardiac maturation as expressed by sarcomeric length (F) and sarcomere alignment (G) in 3D-EHTs containing CM only, CM + sOPTiKD-FN1 EPI without tetracycline, and CM + sOPTiKD-FN1 EPI exposed to tetracycline. (H and I) Frank-Starling curves of active force generation (H) and slopes of active force (I) of 3D-EHTs containing CM only, CM + sOPTiKD-FN1 EPI without tetracycline, and CM + sOPTiKD-FN1 EPI exposed to tetracycline. (J and K) Frank-Starling curves of passive force generation (J) and slopes of passive force (K) of 3D-EHTs containing CM only, CM + sOPTiKD-FN1 EPI without tetracycline, and CM + sOPTiKD-FN1 EPI exposed to tetracycline. (L–O) Ca²⁺ kinetics of 3D-EHTs containing CM only, CM + sOPTiKD-FN1 EPI without tetracycline, and CM + sOPTiKD-FN1 EPI exposed to tetracycline. Each experimental group included N = 3 biological replicates. Mean values; error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.005. Two-sided p values were calculated using a one-way ANOVA with post hoc correction for multiple comparisons.

Figure 5

The addition of rhFN1 increases hPSC-CM maturity in 3D-EHTs without the presence of hPSC-EPI (A) Immunofluorescent imaging of 3D-EHTs shows the arrangement of alpha-actinin within sarcomeres and localization of connexin-43 in constructs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations; sarcomere lengths are quantified in (B). (C) Immunofluorescent imaging of 3D-EHTs highlighting the quantify of FN1 and collagen deposited within constructs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations; FN1 intensities are quantified in (D). (E–H) (E) Frank-Starling curves of active force generation and (F) slopes of active force, as well as (G) curves of passive force and (H) slopes of passive force measured from 3D-EHTs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations. (I–L) Ca²⁺ kinetics of 3D-EHTs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations. Each experimental group included a minimum of N = 3 biological replicates. (I and J) Ca²⁺ kinetics including upstroke velocity and time to peak fluorescence of 3D-EHTs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations. (K and L) Ca2+ kinetics including time to 50% decay and time to 90% decay of 3D-EHTs containing hPSC-CMs, hPSC-CMs + hPSC-EPIs, or hPSC-CMs + rhFN1 at varied concentrations. Mean values; error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.005.

Figure 6

The EPI-CM interactome reveals a regulatory gene network driving cardiac development and regeneration (A) Schematic overview of the study design and experimental procedure. Epicardial secretome and CM membranome were first derived using RNA sequencing. Both datasets were next integrated to generate the EPI-CM interactome, revealing a unique signaling network of regenerative pathways driving cardiac development and tissue repair. All pathways were finally ascribed reparative categories constituting a reference library for cardiac repair. (B) The interactome between hESC-EPI secretome and hESC-CM membranome. Blue nodes on the left represent genes encoding for secreted factors expressed by hESC-EPIs compared with expression in hESC-NC, shown in gray. Orange nodes on the right depict genes encoding membrane-bound receptors expressed by hESC-CM compared with hESC. All binary hESC-EPI to hESC-CM interactions with an experimental score of above 700 in STRINGdb are shown, including a second layer of interactions formed from associations with an experimental score above 400. Respective interactions are shown with gray lines. CEPI-CM regulatory gene network driving cardiac development and regeneration. (C) hESC-EPI-secreted genes are shown as diamond symbols on blue background, and hESC-CM membrane genes are displayed as circles on red background. Interactions with a STRINGdb score of greater than 700 are shown as bold lines and those with a STRINGdb score of less than 700 as fine lines. Strength of gene expression is expressed as L2FC and visualized by the gray tone in hESC-EPI-secreted and hESC-CM membrane gene symbols. The color of the connecting line between two genes describes the reparative category based on a manual literature review with references cited in Table S1. hESC, human embryonic stem cell; hESC-EPI, human embryonic stem cell-derived EPIs; hESC-CM, human embryonic stem cell-derived CMs; hESC-NC, human embryonic stem cell-derived neural crest cells.