Antisenescence Therapy Improves Function in a Human Model of Cardiac Fibrosis-on-a-Chip

Figures

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  Figure 1. A human cardiac fibrosis on a chip model displays a gene signature characteristic of senescence. A, Chip design and seeding. Micromilling is used to create microwells in poly(methyl methacrylate) (PMMA); flexible polydimethylsiloxane (PDMS) rods are suspended across the wells. hiPSC-CMs and cardiac fibroblasts are suspended in a fibrinogen gel and seeded into the wells after thrombin is added to promote fibrin polymerization. B, Schematic of assays. Cardiac fibroblasts cultured in tissue culture plastic were pretreated with 10 ng/mL TGF-β for 48 h (activated) prior to tissue seeding. hiPSC-CMs and activated fibroblasts were combined at a 3:1 ratio in fibrin gel and seeded into the heart-on-a-chip devices, continuously cultured with TGF-β. For healthy controls, hiPSC-CMs were seeded with cardiac fibroblasts not treated with TGF-β, which was also omitted from the cell culture media postseeding. After 3 weeks in culture, RNA was extracted and used for bulk RNA-seq. C, Heat map displaying upregulation of senescence-associated genes in fibrotic tissues: CDKN1A, CDKN2A, IGFBP6, MMP11, SERPINE1, MMP2, IGFBP3, and TIMP2.

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  Figure 2. Treatment with senolytics (D+Q) improves passive tension and active force of contraction in fibrotic tissues. A, Timeline for D+Q treatment of fibrotic tissues. Drug treatment began after 1 week of tissue compaction, without withdrawal of TGF-β to maintain the pathological stimulus. DMSO was used as vehicle control. Force measurements were performed at the end of week 3. B, D+Q treatment significantly improved passive tension (p = 0.0002). C, active force of contraction (p = 0.0185). N = 8, two-tailed Student’s t test.

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  Figure 3. Downregulation of senescence-related genes after D+Q treatment of fibrotic tissues. A, Gene expression assessed by qPCR revealed a significant downregulation of tumor suppressor genes CDKN1A and CDKN2A, as well as the SASP-associated genes IGFBP6 and SERPINE1. GAPDH was used as a housekeeping gene for 2–ΔΔct analysis. N = 5 for CDKN1A (p = 0.0441), IGFBP6 (p = 0.0117), SERPINE1 (p = 0.002). N = 3 for CDKN2A (p = 0.0245). Two-tailed Student’s t test. ΔCt values used for statistical comparisons. B, Human protein cytokine array to determine microtissue secreted factors. Conditioned media from microtissues was assessed for the relative expression level of human cytokines. SASP-related cytokines are highlighted with green boxes. C, The mean pixel density, correlating with expression, is quantified with percent decrease shown for each cytokine. N = 1.

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  Figure 4. Treatment of fibrotic tissues with D+Q does not reduce collagen deposition. A, SHG imaging reveals fibrillar collagen deposition (gray) overlaid with tissue autofluorescence (green). Collagen deposition was quantified by dividing the area of collagen content by the total tissue area. B, Quantification of collagen deposition demonstrated there were no differences between D+Q treated-tissues and vehicle-treated controls (fibrotic), displayed as fold over control. Two-tailed Student’s t test (p = 0.8814). Scale bar = 100 μm.

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  Figure 5. D+Q treatment causes a decrease in the relative fibroblast population and does not alter proliferation. A, Immunohistochemistry of 14 μm-thick cryosections showing cardiomyocytes (green, cTNT+) and fibroblasts (red, vimentin+) counterstained for nuclei (blue, Hoechst). B, Quantification of the percentage of cardiomyocytes and fibroblasts. In fibrotic tissues, the proportion of fibroblasts is ∼24%, whereas it is ∼8% in D+Q-treated tissues (p = 0.0011). Conversely, the proportion of cardiomyocytes increases from ∼76% to ∼ 91% with D+Q treatment (p = 0.0028). Two-tailed Student’s t test. C, In fibrotic tissues, the fibroblast density (cells/area) decreases with D+Q treatment (p = 0.0088). D, Immunohistochemistry to visualize the proportion of actively dividing, ki67-positive nuclei (gray). E, There is no difference in proliferation between fibrotic and D+Q-treated tissues (p = 0.9151). N = 3. Scale bar = 100 μm for all images.