Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis

Abstract

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Keywords: Cardiac fibrosis modelling; Decellularized extracellular matrix; Induced pluripotent stem cells; iPSC-derived-cardiac fibroblasts; iPSC-derived-cardiomyocytes.

Fig. 1

Cardiac fibroblasts derived from express fibroblast- and cardiac-specific markers. (A) Schematic overview of the differentiation protocol used to obtain cardiac fibroblasts from induced pluripotent stem cells. (B) Gene expression analysis of selected pluripotency (blue), fibroblast (green), and cardiac (red) genes in iPSC-derived cardiac fibroblasts as obtained from RT-qPCR. Gene expression values are expressed as fold change (log 2^-ΔΔCt, FC) compared to iPSCs and normalized to iPSCs. Statistical analysis was performed with One-sample t-test (N = 3, n = 3); ** p < 0.01, ***p < 0.001. Data presented as mean ± standard deviation and normalized on GAPDH expression. (C) Representative confocal images of iPSC-derived cardiac fibroblasts stained with antibodies directed against the indicated fibroblast-specific (PDGFR-α, FSP, DDR2, TE-7) and cardiac (GATA4) markers (N = 3, n = 3). The stainings are shown in green and nuclei are counterstained with DAPI (blue) (Scale bar: 50µm). (D) Barplot representation of the percentage of GATA4-positive iPSC-derived cardiac fibroblasts (N = 3, n = 3) obtained from confocal imaging. Data presented as mean ± standard deviation. (E) Representative contour plot images of the expression of the indicated surface markers in iPSC-derived cardiac fibroblasts. (F). Barplot representation of the expression of the indicated markers as obtained from flow cytometry analysis. The values are expressed as percentage of positive cells (N = 3, n = 3). Data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Activation of iPSC-derived cardiac fibroblast to the myofibroblast phenotype. (A) Representative contour plot images of the expression of the indicated markers of activated cardiac fibroblasts in ctrl (left), pro- (middle) and anti-fibrotic (right) iPSC-derived cardiac fibroblasts. (B) Violin plot representation of the expression of the indicated markers in iPSC-derived cardiac fibroblasts. Values are indicated as percentage of α-SMA/FAP double-positive cells in ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts (N = 3, n = 4). **= p < 0.01. Data presented as mean ± standard deviation. (C) Violin plot representation of the expression of the indicated markers in iPSC-derived cardiac fibroblasts. Values are indicated as log 2^-ΔΔCt; fold change compared to ctrl (N = 3, n = 3) and normalized on GAPDH expression. One-way ANOVA followed by Kruskal-Wallis test, * p < 0.05. Data presented as mean ± standard deviation. (D) Representative super-resolution microscopy images of focal adhesion protein vinculin expression (green) in ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts. Nuclei are counterstained with DAPI (blue) (Scale bar: 10µm). (E) Violin plot representation of the area of single ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts (N = 3; n = 19 for control, n = 17 for pro-fibrotic, n = 19 for anti-fibrotic). One-way ANOVA followed by Kruskal-Wallis test. ** p < 0.01, **** p < 0.0001. Data presented as mean ± standard deviation. (F) Violin plot representation of the number of focal adhesion (FA) per cell (left) (N = 3; n = 15 for control, n = 16 for pro-fibrotic, n = 18 for anti-fibrotic) in iPSC-derived cardiac fibroblasts. Violin plot representation of the area of focal adhesions (right) (N = 3; n = 584 for control, n = 364 for pro-fibrotic, n = 135 for anti-fibrotic) in iPSC-derived cardiac fibroblasts. One-way ANOVA followed by Kruskal-Wallis test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Data presented as mean ± standard deviation. (G) Representative confocal microscopy image of α-SMA (green) and F-actin (grey) in ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts. Nuclei are counterstained with DAPI (blue) (Scale bar: 50µm). (H) Violin plot representation of stress fibres coherency in ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts as obtained by confocal image analysis. The values are expressed as arbitrary units (a.u.). One-way ANOVA followed by Kruskal-Wallis test (N = 3, n = 4), *= p < 0.05. Data presented as mean ± standard deviation. (I) Violin plot representation of TGF-β1 concentration in the supernatant of ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts. The values are normalized to cell count. One-way ANOVA followed by Kruskal-Wallis test (N = 4, n = 4), * p < 0.05. Data presented as mean ± standard deviation. (J) Representative confocal images of ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts stained for SMAD2/3 (red) and F-ACTIN (grey). The nuclei are counterstained with DAPI (blue). (Scale bar: 30µm). (K) Violin plot representation of the percentage of ctrl, pro- and anti-fibrotic iPSC-derived cardiac fibroblasts found positive for nuclear SMAD 2/3 in confocal microscopy imaging (N = 3; n = 4 for control, n = 5 for pro-fibrotic, n = 6 for anti-fibrotic). One-way ANOVA followed by Kruskal-Wallis test, * p < 0.05. Data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The extracellular matrix deposited by activated cardiac fibroblasts shows a fibrotic signature. (A) Representative scanning electron microscopy images showing the architecture of the dECMs deposited by cardiac fibroblasts obtained from iPSCs and exposed to ctrl, pro- and anti-fibrotic culture conditions (Scale bar: 5µm). (B) Representative orthogonal view of dECMs derived from ctrl, pro-, and anti-fibrotic iPSC-derived cardiac fibroblasts stained for collagen I (red). The images were obtained by z-stack confocal pictures. (C) Representative confocal images of the expression of the indicated proteins (green) in dECMs deposited by ctrl, pro-, and anti-fibrotic iPSC-derived cardiac fibroblasts (Scale bar: 50 µm). (D) Heatmap representation of the matrisome proteins found in dECMs obtained from ctrl, pro-, and anti-fibrotic iPSC-derived cardiac fibroblasts and clustered according to their expression pattern. The colour code is adjusted row-by-row based on the row Z-score values (-2 ≤ Z-score ≤ 2). (E) Principal component analysis of the clustering of dECMs deposited by ctrl, pro-, and anti-fibrotic iPSC-derived cardiac fibroblasts based on the expression of matrisome proteins. Key components of the matrisome driving the clustering of each of the categories are shown. The length of the arrows weighs the contribution of each protein (N = 3). (F) Volcano plot representation of the proteins regulated proteins in pro-fibrotic compared to ctrl dECM. The proteins significantly upregulated are shown in green, while those downregulated are indicated in red colour (padj < 0.05, log2Fc > ǀ1ǀ). (G) Venn diagram representation of the relative abundance of proteins belonging to the indicated sub-categories of the matrisome found differentially regulated in ctrl vs pro-fibrotic dECM. (H) Barplot representation of the indicated Gene Ontology categories (Biological Processes, GO:0030198; GO:0030199; GO:0043062; GO;0045229) found significantly regulated in pro-fibrotic vs. ctrl dECM based on the differential expression of matrisome proteins. The graph was obtained from ENRICHR database (padj < 0.05, log2Fc > ǀ1ǀ). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Micromechanics analysis of the dECMs deposited by iPSC-derived cardiac fibroblasts. (A) Representative colour-coded maps showing the surface topography of the dECMs deposited by ctrl (left), pro- (middle) and anti-fibrotic (right) iPSC-derived cardiac fibroblasts. The colour code is meant to highlight dECM surface irregularities. (B) Violin plot representation of Young's Modulus values (Pa: Pascal) obtained for ctrl, pro-, and anti-fibrotic dECMs by nanoindentation. (N = 3, n≥30). One-way ANOVA followed by Kruskal-Wallis test. **** p < 0.0001. Data presented as mean ± standard deviation. (C) Violin plot representation of the viscoelastic modulus (Damping factor: tanδ) as obtained after indentation of ctrl and pro-fibrotic dECMs at the indicated frequencies (Hertz: Hz). Multiple t-test validated by Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (N = 3, n = 3) ** p < 0.01, **** p < 0.0001. Data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Pro-fibrotic dECM improve iPSC-CMs survival. (A) Representative confocal images of iPSC-derived cardiomyocytes cultured for 10 days on ctrl or pro-fibrotic dECMs and stained for α-actinin (green) and collagen IV (Col IV, red). Nuclei were counterstained with DAPI (blue) (scale bar: 20µm). (B) Histogram representation (left) and relative violin plot quantification (right) of the percentage of cTnT+ cells found in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECM, as obtained by flow cytometry analysis. Ratio paired t-test (N = 4, n = 4). ** p < 0.01, data presented as mean ± standard deviation. (C) Representative confocal images (left) of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECM and stained for α-actinin (green), YAP (red) and Col IV (grey). Cell nuclei were counterstained with DAPI (blue) (Scale bar: 30 µm). Violin plot representation (right) of YAP nucleus/cytoplasm ratio in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs. The values are indicated as arbitrary unit (a.u.). Non-parametric Mann-Whitney test (N = 2; n = 130 for ctrl, n = 124 for pro-fibrotic). ** p < 0.01. Data presented as mean ± standard deviation. (D) Violin plot representation of NFATC1 nucleus/cytoplasm ratio in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs. The values are indicated as arbitrary unit (a.u.). Non-parametric Mann-Whitney test (N = 2; n = 29 for control, n = 25 for pro-fibrotic). N.S.=non-significant, data presented as mean ± standard deviation. (E) Representative confocal images of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs and stained for α-actinin (red) and Ki67 (green). Nuclei were counterstained with DAPI (blue). (F) Violin plot representation of the co-expression of cTnT and Ki67 in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs as obtained from flow cytometry analysis. The values are expressed as percentage of double positive cells. Ratio paired t-test (N = 3, n = 3). N.S.= non-significant, data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Pro-fibrotic dECM affects iPSC-derived cardiomyocyte sarcomere organization. (A) Violin plot representation of the projected area of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 383 for control, n = 313 for pro-fibrotic) (B) Violin plot representation of the projected nuclear area of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 336 for control, n = 291 for pro-fibrotic). (C) Violin plot representation of the circularity of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs. Non-parametric Mann-Whitney t-test (N = 3; n = 415 for control, n = 342 for pro-fibrotic). N.S.=non-significant, *** p < 0.001. Data presented as mean ± standard deviation. (D) Representative confocal images (left) of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs and decorated with α-actinin (green), MRTF-A (red) and collagen IV (white). The nuclei were counterstained with DAPI (blue). (Scale bar: 30 µm). Violin plot representation of MRTF-A nucleus/cytoplasm ratio (right) in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs. The values are expressed as arbitrary units (a.u.). Non-parametric Mann-Whitney t-test (N = 2; n = 139 for control, n = 94 for pro-fibrotic). **** p < 0.0001, data presented as mean ± standard deviation. (E) Representative confocal images of iPSC-derived cardiomyocytes differentiated for 20 days, cultured for 10 additional days on either ctrl or pro-fibrotic dECMs and stained for α-actinin (white). (F) Analysis of the percentage of cardiomyocytes characterized by assembled sarcomere following the interaction with either control or pro-fibrotic dECM. Non-parametric Mann-Whitney t-test (N = 3; n = 8). *= p < 0.05. Data presented as mean ± standard deviation. (G) Violin plot representation of the sarcomere length of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 9 for control, n = 10 for pro-fibrotic). Non-parametric Mann-Whitney t-test. N.S.=non-significative. Data presented as mean ± standard deviation. (H) Violin plot representation of sarcomere homogeneity in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 10 for control, n = 9 for pro-fibrotic). Non-parametric Mann-Whitney t-test. N.S.=non-significative. Data presented as mean ± standard deviation. (I) Violin plot representation of the sarcomere organization score in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 10). Non-parametric Mann-Whitney t-test. N.S.=non-significative. Data presented as mean ± standard deviation. (J) Violin plot representation of the sarcomere alignment in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 9 for control, n = 10 for pro-fibrotic). Non-parametric Mann-Whitney t-test. N.S.=non-significative. Data presented as mean ± standard deviation. (K) Violin plot representation of the sarcomere uniformity in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3; n = 7 for control, n = 9 for pro-fibrotic). Non-parametric Mann-Whitney t-test. ** p < 0.01. Data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Pro-fibrotic dECM influences iPSC-derived cardiomyocytes calcium handling. (A) Violin plot representation of the beating rate of iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs. Non-parametric Mann-Whitney t-test (N = 3; n = 27 for control, n = 19 for pro-fibrotic). N.S.=non-significant, data presented as mean ± standard deviation. (B) Violin plot representation of calcium decay time (dT80-dT50-dT30) in iPSC-derived cardiomyocytes differentiated for 20 days and cultured for 10 additional days on either ctrl or pro-fibrotic dECMs (N = 3. dt80: n = 30 for control, n = 28 for pro-fibrotic; dt50: n = 30 for control, n = 27 for pro-fibrotic; dt30: n = 30 for control, n = 28 for pro-fibrotic). Non-parametric Mann-Whitney t-test. N.S.=non-significant; * p < 0.05, ** p < 0.01. Data presented as mean ± standard deviation. (C) Representative traces of Ca2+ transients (CaT) recorded from iPSC-CMs-2 on ctrl (black) and pro-fibrotic cFbs-dECM-2 (red). (D) Violin plot representing the decay time (dT80-dT50-dT30) in iPSC-CMs-2 cultured on ctrl and pro-fibrotic dECM-2 (N = 4. dt80: n = 40; dt50: n = 37; dt30: n = 40). Statistical analysis performed by non-parametric Mann-Whitney t-test. * p < 0.05. Data presented as mean ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)