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Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.

ABSTRACT: Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.

SUBMITTER: Mantri M

PROVIDER: S-EPMC7979764 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AIMS:Fibroblast to myofibroblast trans-differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de-differentiation could mitigate CF-related pathologies. We determined whether 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors-statins, commonly prescribed to patients at risk of heart failure (HF)-can de-differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. METHODS AND RESULTS:Either in vitro statin treatment of differentiated myofibroblasts (n = 3-6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4-12). In vitro, hVFs were differentiated by transforming growth factor-β1 (TGF-β1) for 72 h (TGF-72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α-smooth muscle actin (α-SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration-dependently de-differentiated the myofibroblasts. The respective half-maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 μmol/L), the reduced product of HMG-CoA, prevented the statin-induced de-differentiation (α-SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 μmol/L), a cholesterol synthesis-independent HMG-CoA reductase pathway intermediate, completely prevented the statin-induced de-differentiation (α-SMA/GAPDH ratios: 0.89 ± 0.05 [TGF-72 h + 72 h], 0.63 ± 0.02 [TGF-72 h + simvastatin], and 1.2 ± 0.08 [TGF-72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co-incubation of simvastatin (200 nmol/L) with glibenclamide (10 μmol/L), a KATP channel inhibitor, attenuated the simvastatin-induced de-differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de-differentiation effect (0.33 ± 0.02). OCR (pmol O2 /min/μg protein) was significantly decreased in the simvastatin-treated hVFs, including basal (P = 0.002), ATP-linked (P = 0.01), proton leak-linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP-linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (α-SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01). CONCLUSIONS:This study demonstrates the de-differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of KATP channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.

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Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.

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