Cardiac Fibrotic Remodeling on a Chip with Dynamic Mechanical Stimulation

Abstract

Cardiac tissue is characterized by being dynamic and contractile, imparting the important role of biomechanical cues in the regulation of normal physiological activity or pathological remodeling. However, the dynamic mechanical tension ability also varies due to extracellular matrix remodeling in fibrosis, accompanied with the phenotypic transition from cardiac fibroblasts (CFs) to myofibroblasts. It is hypothesized that the dynamic mechanical tension ability regulates cardiac phenotypic transition within fibrosis in a strain-mediated manner. In this study, a microdevice that is able to simultaneously and accurately mimic the biomechanical properties of the cardiac physiological and pathological microenvironment is developed. The microdevice can apply cyclic compressions with gradient magnitudes (5-20%) and tunable frequency onto gelatin methacryloyl (GelMA) hydrogels laden with CFs, and also enables the integration of cytokines. The strain-response correlations between mechanical compression and CFs spreading, and proliferation and fibrotic phenotype remolding, are investigated. Results reveal that mechanical compression plays a crucial role in the CFs phenotypic transition, depending on the strain of mechanical load and myofibroblast maturity of CFs encapsulated in GelMA hydrogels. The results provide evidence regarding the strain-response correlation of mechanical stimulation in CFs phenotypic remodeling, which can be used to develop new preventive or therapeutic strategies for cardiac fibrosis.

Keywords: cardiac fibrosis; hydrogels; mechanical stimulation; organ-on-a-chip; transforming growth factor-β.

Figure 1.

Activation of CFs towards cardiac myofibroblast phenotypes. Interstitial fibroblasts are characterized by the production of fibronectin and the absence of filamentous-actin, α-SMA, and ED-A fibronectin. Under mechanical stress or inflammatory cytokines, fibroblasts are activated into proto-myofibroblasts. The proto-myofibroblast produces ED-A fibronectin, contains stress fibers and focal adhesions, but does not yet contain the contractile α-SMA thus representing an immature myofibroblast. The increasing mechanical stress and TGF-β promote the modulation of proto-myofibroblasts into mature myofibroblasts. Mature myofibroblasts show abundant production of ED-A fibronectin and F-actin and are characterized by the presence of α-SMA. The transition from fibroblasts to proto-myofibroblasts is reversible.

Figure 2.

(a) Schematic illustration of bioreactor fabrication consisting of N₂ pressure chamber, PDMS membrane with pillars, medium chamber and TMSPMA coated glass from the bottom up. (b) Flowchart of bioreactor assembly, loading CFs with GelMA pre-polymer solution, UV exposure to cure cell-laden gels along PDMS pillars. (c) Side view of five hydrogels (Diameter: 3 mm) patterned on top of pillars in the bioreactor and sequential photographs showing the compression and relaxation of a patterned hydrogel under cyclic compression. Scale bar = 4 mm. Green dye was incorporated into the hydrogel to aid in visualization. (d) Schematic illustration of bioreactors in a static state (top) and actuated state (bottom) by injection of gas to generate a different strain of compression upon hydrogels, the hydrogels were normalized with numbers 1–5 indicating cavity diameter 8–5 mm of gas chambers. (e-f) Experimental data of compression strainas a function of hydrogel position and applied N₂ pressures at 1 Hz in (e) air and (f) culture medium that filled inside the bioreactor leaving the medium tubes open (n=5). (g) Characterization of the dynamic compression response of a patterned hydrogel upon actuation of a post by applied pressure. (h) Young’s modulus of hydrogels after different incubation days of mechanical compression under 1 Hz actuation. (n=5)

Figure 3.

(a) Representative confocal images of EdU Click-iT labeling (green) of CF-laden 3D GelMA hydrogels with different compression strains at days 1, 4, and 7. (b) Representative quantification of proliferating cells inside CF-laden 3D GelMA hydrogels as determined by the percentage of EdU positive cells at days 1, 4, and 7 of culture (n=6). CFs cultured on 2D well plates were taken as control (n=6). (c and d) CFsspreading area and aspect ratio after 7 days of mechanical stimulation(n=8). (e) Representative confocal images of Actin/DAPI results under different compression strain from 0 to 20%. Data depict Mean ± Standard deviation. *p<0.05.

Figure 4.

RT-PCR of relative mRNA expression of α-SMA, Col-I, TGF-β, Fn and MMP-2 of (a) CFs of P2 or P5 which were cultured on 2D TCPS cell plate or in 3D hydrogels in microdevices; (b) CFs of P2 cultured in 3D GelMA hydrogels after 7 days cyclic compression stimulation of different strain from 0 to 20% (n=4). Data depicts fold-change ± standard deviation. *p<0.05.

Figure 5.

A cardiac fibrosis model was established by treating CFs (P5) in the microdevices with 10 ng/mL TGF-β1 for 24 hours prior to compression stimulation, the first group receiving only mechanical stimulation, labeled M; the second group continuously treated with TGF-β inhibiting drug, Tranilast, and mechanical stimulation, labeled DM; the third group continuously treated with TGF-β1 and mechanical stimulation for 7 days, labeled TM; and the last group without any mechanical compression or TGF-β1 treatment, labeled C. (a) Representative confocal images of immunofluorescence stained α-SMA (green) and collagen-I (Col I, red) after 7 days cyclic compression stimulation with different strains from 0 to 20% (n=4). (b) RT-PCR of relative mRNA expression of α-SMA, Col-I, Fnand MMP-2 ofCFsafter 7 days cyclic compression stimulation of different strain from 0 to 20% (n=4). Data depicts fold-change ± standard deviation. *p<0.05,** p<0.01, *** p<0.001.