Targeting Interleukin-8–Mediated Cellular Crosstalk Reverses Hypertrophic Cardiomyopathy and Cardiac Fibrosis in Noonan Syndrome

Figures

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  Figure 1.. Biallelic LZTR1 deficiency in cardiomyocytes (CMs) and non-CMs induces RAS GTPase accumulation. A, Representative Masson trichrome-stained section of right ventricular myocardium from a patient with Noonan syndrome with LZTR1 deficiency (extracellular matrix, blue; CM, red). B, Schematic of RAS-MAPK signaling in the context of LZTR1 deficiency. C, Generation of LZTR1-deficient induced pluripotent stem cells (iPSCs) with heterozygous and homozygous truncating variants using CRISPR-Cas9 and directed differentiation of iPSCs into iPSC-CMs, iPSC-cardiac fibroblasts (CFs), and iPSC-endothelial cells (ECs). D, Representative immunofluorescence images of LZTR1WT/WT and LZTR1KO/KO iPSC-CMs stained for α-actinin (orange), iPSC-CFs stained for COL1A1 (green), and iPSC-ECs stained for PECAM1 (magenta); nuclei were counterstained with Hoechst 33342 (white). Scale bar: 50 µm. E, Representative Western blot images of LZTR1WT/WT and LZTR1KO/KO iPSC-CMs, iPSC-CFs, and iPSC-ECs of LZTR1, RAS GTPases, and cell type–specific markers (cTNT, COL1A1, and CD144); vinculin is shown as housekeeper. F through H, Quantitative analysis of Western blots for MRAS (F), RIT1 (G), and pan-RAS recognizing HRAS,…

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  Figure 2.. LZTR1-deficient cardiac fibroblasts (CFs) promote cellular hypertrophy in cardiomyocytes (CMs). A, LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO induced pluripotent stem cell (iPSC)-CMs were analyzed by quantitative global proteomics via LC-MS/MS on day 60 of differentiation. n=5 independent differentiations per iPSC line from LZTR1WT/WT, LZTR1Ex1-KO/WT, LZTR1Ex1-KO/Ex1-KO, LZTR1Ex17-KO/WT, and LZTR1Ex17-KO/Ex17-KO, measured in technical duplicates. B, Volcano plot comparing the global proteome of LZTR1-deficient iPSC-CMs (LZTR1Ex1-KO/Ex1-KO and LZTR1Ex17-KO/Ex17-KO) with functional LZTR1 samples (LZTR1WT/WT, LZTR1Ex1-KO/WT, and LZTR1Ex17-KO/WT). RAS proteins are highlighted. Differential abundance was assessed using a t test with Benjamini-Hochberg multiple testing correction. The dotted line indicates an adjusted P value of 0.05. C, Reactome pathway enrichment analysis in LZTR1-deficient vs functional LZTR1 iPSC-CMs. Node color indicates up- and downregulated proteins within each pathway. Node size reflects adjusted P value. D, Relative (Rel) transcript levels of the hypertrophy-associated genes NPPA and NPPB, assessed by RT-qPCR in LZTR1WT/WT and LZTR1KO/KO iPSC-CMs. Data were…

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  Figure 3.. LZTR1-deficient cardiac fibroblasts (CFs) show RIT1-dependent cell cycle activity. A, LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO iPSC-CFs were analyzed by quantitative global proteomics via LC-MS/MS on day 30 of differentiation. n=3 independent differentiations per induced pluripotent stem cell (iPSC) line from LZTR1WT/WT, LZTR1Ex1-KO/WT, LZTR1Ex1-KO/Ex1-KO, LZTR1Ex17-KO/WT, and LZTR1Ex17-KO/Ex17-KO, measured in technical duplicates. B, Volcano plot comparing the global proteome of LZTR1-deficient iPSC-CFs (LZTR1Ex1-KO/Ex1-KO and LZTR1Ex17-KO/Ex17-KO) with functional LZTR1 samples (LZTR1WT/WT, LZTR1Ex1-KO/WT, and LZTR1Ex17-KO/WT). RAS proteins are highlighted. Differential abundance was assessed using a t test with Benjamini-Hochberg multiple testing correction. The dotted line indicates an adjusted P value of 0.05. C, Reactome pathway enrichment analysis in LZTR1-deficient vs functional LZTR1 iPSC-CFs. Node color indicates up- and downregulated proteins within each pathway. Node size reflects adjusted P value. D, Representative Ki-67 immunofluorescence images (orange) of LZTR1WT/WT and LZTR1KO/KO iPSC-CFs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 50 µm. E,…

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  Figure 4.. LZTR1-deficient engineered connective tissues exhibit a fibrotic signature. A, LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO induced pluripotent stem cell (iPSC)-cardiac fibroblasts (CFs) were cast with a collagen matrix around poles for generation of engineered connective tissues (ECTs), and tissues were subsequently analyzed for tissue stiffness and transcriptomic signatures. B, Pole deflection of LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO ECTs were determined daily over 13 days of culture. Pole distances were normalized to day 0 of tissue culture. n=26 to 39 tissues from 2 to 3 independent differentiations per iPSC line from LZTR1WT/WT, LZTR1Ex1-KO/WT, and LZTR1Ex1-KO/Ex1-KO. C, Representative macroscopic images of LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO ECTs on day 13 after casting. Scale bar: 1 mm. D, Cross-sectional area of LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO ECTs was calculated based on imaging top and side views. n=28 to 36 tissues from 3 independent differentiations per iPSC line from LZTR1WT/WT, LZTR1Ex1-KO/WT, and LZTR1Ex1-KO/Ex1-KO. E and F, Biomechanical properties of LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO ECTs were determined by stress-strain analysis, and Young’s modulus…

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  Figure 5.. LZTR1-deficient cardiac fibroblasts (CFs) display RIT1-dependent impaired IL-8 (interleukin-8) signaling. A, Conditioned (cond) media from LZTR1WT/WT and LZTR1KO/KO induced pluripotent stem cell (iPSC)-CFs were collected and analyzed using a cytokine array for detection of more than 100 cytokines, chemokines, and growth factors. B, Representative cytokine array membranes incubated with conditioned media from LZTR1WT/WT and LZTR1KO/KO iPSC-CFs; highly secreted factors are highlighted. C, Quantitative analysis of secreted cytokines in conditioned media from LZTR1WT/WT and LZTR1KO/KO iPSC-CFs. Data were normalized to the assay’s positive controls. n=2 independent differentiations per iPSC line from LZTR1WT/WT and LZTR1Ex1-KO/Ex1-KO, measured in technical duplicates. D through F, Relative transcript levels of CXCL8 (D) and its receptors CXCR1 (E) and CXCR2 (F), assessed by RT-qPCR in LZTR1WT/WT, LZTR1KO/WT, and LZTR1KO/KO iPSC-cardiomyocytes (CMs), iPSC-CFs, and engineered connective tissues (ECTs). Data were normalized to housekeepers (GAPDH, TUBB5, and RPL37A) and corresponding WT. For CMs and CFs, n=6 independent differentiations per iPSC line from LZTR1WT/WT, LZTR1Ex1-KO/WT,…

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  Figure 6.. IL-8 (interleukin-8)–CXCR1 signaling promotes profibrotic and hypertrophic remodeling. A, Induced pluripotent stem cell (iPSC)-cardiac fibroblasts (CFs), engineered connective tissues (ECTs) and iPSC-cardiomyocytes (CMs) were stimulated with recombinant IL-8 and subsequently analyzed for its influence on proliferation, tissue stiffness, collagen secretion, and cellular hypertrophy. B and C, Proliferation growth curves (solid lines, mean; shaded areas, mean±SEM) (B) and quantitative analysis (C) for LZTR1WT/WT iPSC-CFs under stimulation with 100 ng/mL IL-8 or vehicle control; quantification of proliferation was determined by area under the curve (AUC) analysis and normalized to vehicle-treated samples; n=16 samples per condition from 4 independent differentiations from LZTR1WT/WT. D, Pole deflection of LZTR1WT/WT ECTs under stimulation with 100 ng/mL IL-8 or vehicle control was determined daily over 11 days of culture. Pole distances were normalized to day 0 of tissue culture. n=5 to 6 tissues per condition from LZTR1WT/WT. E, Representative fluorescence images of collagen reporter iPSC-CFs with fluorescently tagged COL1A2, showing collagen deposition under IL-8 stimulation over…

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  Figure 7.. Inhibition of IL-8 (interleukin-8)–CXCR1 signaling reverses LZTR1-associated cardiac remodeling. A, LZTR1WT/WT and LZTR1KO/KO induced pluripotent stem cell (iPSC)-cardiac fibroblasts (CFs), engineered connective tissues (ECTs), and iPSC-cardiomyocytes (CMs) were treated with the IL-8 signaling inhibitor reparixin and evaluated for drug effects on proliferation, tissue stiffness, fibrotic signatures, signaling activity, and cellular hypertrophy. B and C, Proliferation growth curves (solid lines, mean; shaded areas, mean±SEM) (B) and quantitative analysis (C) for LZTR1KO/KO iPSC-CFs under treatment with 10 µM reparixin or vehicle control. Quantification of proliferation was determined by area under the curve (AUC) analysis and normalized to vehicle-treated samples. n=23 to 24 samples per condition from 4 independent differentiations from LZTR1Ex1-KO/Ex1-KO. D, Pole deflection of LZTR1WT/WT and LZTR1KO/KO ECTs under treatment with 10 µM reparixin or vehicle control was determined daily over 11 days of culture with treatment initiated on day 5. Pole distances were normalized to day 0 of tissue culture. Shown is a representative treatment round with 6 to 9 tissues per condition from…

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  Figure 8.. Aberrant IL-8 (interleukin-8)–CXCR1 signaling represents a shared pathogenic mechanism driving cardiac pathology in Noonan syndrome. A, Induced pluripotent stem cells (iPSCs) derived from a patient with Noonan syndrome carrying biallelic LZTR1, RIT1F99L, RAF1S257L, or MRASG23V variants and corresponding CRISPR-corrected (corr) isogenic controls were differentiated into iPSC-cardiac fibroblasts (CFs) and analyzed for cytokine secretion, profibrotic gene expression, and proliferation under basal conditions and after reparixin treatment. B and C, Representative cytokine array membranes incubated with conditioned media from LZTR1biallelic and LZTR1biallelic-corr (B) and from RIT1F99L and RITF99L-corr iPSC-CFs (C); highly secreted factors are depicted. D, Quantitative analysis of secreted IL-8 in conditioned media from LZTR1biallelic, RIT1F99L, RAF1S257L iPSC-CFs, their respective CRISPR-corrected controls, as well as MRASG23V and WT. Data were normalized to the assay’s positive controls. n=2 independent differentiations per iPSC line from LZTR1biallelic, LZTR1biallelic-corr, RIT1F99L, RIT1F99L-corr, RAF1S257L, RAF1S257L-corr, MRASG23V and WT, measured in technical duplicates. E…