In Vitro Mechanical Stimulation to Reproduce the Pathological Hallmarks of Human Cardiac Fibrosis on a Beating Chip and Predict The Efficacy of Drugs and Advanced Therapies

Figure 1. Fibrotic human cardiac model development: a) uBeat Stretch Platform encompassing 3 biologically independent cell culture chambers (red, yellow, blue) with a common actuation line; b) Working principle of the uBeat technology: the cell culture chamber houses two rows of hanging shield‐shaped pillars that i) confine a 3D microtissue (i.e., cell‐laden hydrogel) flanked by two lateral medium channels (top view) and ii) enable the application of 10% uniaxial strain to the cultured microtissues (lateral view); c) Experimental plan followed to achieve the generation of the models.

Figure 2. Fibroblast to myofibroblast transition in the human cardiac model subjected to chemical (TGF‐β1) and/or mechanical stimulations: a) Representative immunofluorescence images of myofibroblasts, identified by a‐SMA staining (green); b) Quantification of α‐SMA positive cells as percentage of the total number of cells in the microtissue. Data are represented as Mean ± SD (n = 6). Cardiac fibrosis markers in the human models among the four different experimental groups: mRNA level expression normalized to GAPDH of c) Tgfβ1, d) Collagen type‐I (Col1a1), e) Collagen type‐III (Col3a1), and f) their ratio (Col3a1/Col1a1) (data calculated with 2‐ΔCt method). Scalebar 100 µm. Data are represented as mean ± SD (n >= 3). Kruskal‐Wallis analysis of variance and Dunn's post‐test (* = p < 0.05, ** = p < 0.01).

Figure 3. ECM components deposition in the human cardiac model after 7 days of culture in the four different experimental groups: a) Representative images of immunofluorescent staining for Collagen I (yellow), Aggrecan (Purple) and Fibronectin (red); b) Quantification of Collagen I, Aggrecan, and Fibronectin production, calculated as the ratio between the percentage of stained area and the total number of cell nuclei. Scalebar 100 µm for Collagen I and Aggrecan and 30 µm for Fibronectin. Data are represented as mean ± SD (n >= 3). Kruskal‐Wallis analysis of variance and Dunn's post‐test (* = p < 0.05). c) Droplet digital PCR analysis of RhoA/ROCK2 and Hippo pathway‐associated genes in the human models among the four different experimental groups: mRNA level expression normalized to GAPDH of i) RhoA, ii) ROCK2, iii) YAP1, iv) TAZ, v) TEAD, vi) CTGF, vii) PAI‐1, viii) GSK3b, ix) b‐catenin, x) TGFb‐R1, xi) TGFb‐R2, xii) NPRA and xiii) BNP. Data are represented as mean ± SD (n >= 3). Kruskal‐Wallis analysis of variance and Dunn's post‐test (* = p < 0.05).

Figure 4. Effect of drugs in preventing the fibrotic traits elicited by the mechanical stimulation in the uScar human model: a) Schematic representation of the experimental plan; b) Representative immunofluorescence images of activated fibroblasts (α‐SMA‐green) and ECM components, such as Collagen I (yellow), Aggrecan (purple) and Fibronectin (Red) and c) relative quantification when Pirfenidone is administered (horizontal dotted line refers to the static controls); d) phenotype switch and ECM deposition quantification when Tranilast is administered. The ECM component quantification was derived as the ratio between the percentage of stained area and the total number of cell nuclei. Scale bar:100 µm. Data are represented as mean ± SD. Kruskal‐Wallis analysis of variance with Dunn's post‐test for all the data, except for α‐SMA with Pirfenidone, analyzed by Ordinary one‐way Anova multiple comparison test after verifying the normality of the dataset.

Figure 5. Study of miRcombo‐mediated direct reprogramming of AHCFs into iCMs with/without cardiac tissue‐like cyclic mechanical stimulation in the uScar human model: a) Schematic representation of the experimental plan; b) mRNA level expression of cardiomyocyte typical markers (TNNT2, MYL7) in static samples obtained from AHCFs transfected with negmiR or miRcombo after 15 days of culture; c) mRNA level expression of TNNT2, MYL7 and fibrotic marker COL1A2 in static or dynamic samples obtained from AHCFs transfected with miRcombo after 15 days of culture; Representative immunofluorescence images of d) a‐sarcomeric actinin cardiomyocytes‐related protein (red) with DAPI counterstaining (blue) and of e) Collagen I fibrosis marker (purple) with relative quantification in static and dynamic samples obtained from AHCFs transfected with miRcombo after 15 days of culture. Scalebar 50 µm for a‐Sarcomeric Actinin and 100 µm for Collagen I. Data are presented as mean ± SEM. Statistical differences between the groups were determined with two‐sided t‐tests.

Figure 6. Effect of the advanced gene therapy in reverting the fibrotic traits elicited by the mechanical stimulation in the uScar human model: a) Schematic representation of the experimental plan; b) Representative immunofluorescence images of α‐SMA (green), collagen I (purple), α‐Sarcomeric Actinin (red), DDR2 (light blue) and DAPI counterstaining (blue) in the four experimental groups and c) relative quantifications. Scalebar 100 µm. Data are represented as mean ± SD. Kruskall‐Wallis analyses of variance with Dunn's post‐test was used (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).