佐藤 滋

Cardiac fibroblasts (CFs) are the predominant non-myocyte cell type in the heart and play central roles in extracellular matrix remodeling and intercellular signaling during cardiac physiology and pathology. However, the bioenergetic basis underlying CF functions remains poorly understood, mainly due to the lack of tools for visualizing intracellular adenosine triphosphate (ATP) dynamics with high spatiotemporal resolution. Here, we established immortalized murine cardiac fibroblasts stably expressing the genetically encoded ATP indicator GO-ATeam2 based on Förster Resonance Energy Transfer (FRET). The resulting CF7/GO-ATeam2 cell line allows real-time and quantitative monitoring of cytosolic ATP levels in living cells. CF7/GO-ATeam2 cells exhibited robust proliferation and quick responses to change of cytosolic ATP level. We demonstrated dynamic cytosolic ATP imaging upon pharmacological perturbations of oxidative phosphorylation and glycolysis, as well as under growth factor stimulation. Our work provides the CF7/GO-ATeam2 platform, a versatile cellular resource for dissecting the metabolic regulation of cardiac fibroblasts, offering new opportunities to explore energy dynamics in cardiac physiology and disease.

AIMS: Hypoxia-inducible factor (HIF) signalling influences cardiomyocyte differentiation, maturation, and metabolic adaptation under pathological conditions. HIF-Prolyl hydroxylase domain (HIF-PH) inhibitors, which target this pathway, have been introduced for the treatment of renal anaemia. Their precise effect or safety on cardiac function remains unclear because their pharmacokinetics and distribution are not well-understood. This study aimed to examine HIF signalling activation in adult cardiomyocytes (CMs). METHODS AND RESULTS: We used tamoxifen (TAM)-inducible, CM-specific von Hippel-Lindau (VHL) knockout (VHL-MCM) mice to activate CM HIF signalling. Then we subjected the mice to normal ageing or high-fat diet (HFD) and L-NAME feeding, a murine model of heart failure with preserved ejection fraction (HFpEF). In normal ageing group, there was no difference in the echocardiographic parameters or tissue fibrosis between VHL-MCM and control mice. VHL-MCM mice exhibited significantly increased capillary density and higher expression levels of HIF-target genes (P = 0.0248, two-way ANOVA). Under HFD + L-NAME treatment, VHL-MCM mice showed transient but significantly preserved global longitudinal strain (GLS) at 12 weeks post-TAM injection compared to controls (P = 0.0284, two-way ANOVA). Sirius red staining indicated a trend towards reduced whole-heart and interstitial fibrosis with significant increase in capillary density in VHL-MCM mice. CONCLUSION: Sustained HIF signalling activation in adult CM does not impair the cardiac structure and function in normal ageing process and shows transient yet beneficial effect in murine HFpEF model.

Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in cellular and tissue adaptation to low oxygen conditions. Although inflammatory stimuli such as lipopolysaccharide (LPS) also increase HIF-1α levels under normoxia, its transcriptional activity and regulatory mechanisms in this context remain unclear. To address this, we performed chromatin immunoprecipitation sequencing and transcriptome analyses in murine macrophages stimulated with either LPS or hypoxia. Both stimuli stabilized HIF-1α protein but via distinct mechanisms: hypoxia acted post-translationally, whereas LPS increased Hif-1α mRNA expression. Genome-wide HIF-1α binding was observed under both conditions; however, only hypoxia induced broad transcriptional activation of target genes, whereas LPS upregulated a restricted set, mostly glycolytic genes. Motif enrichment analysis revealed that hypoxia, but not LPS, promoted cooperative transcription factor engagement, including HIF-1β, ETS, and bZIP family members. Hypoxia also increased H3K27 acetylation at HIF-1α target loci, consistent with a transcriptionally permissive chromatin state. In contrast, LPS led to reduced H3K27ac at noninduced loci, suggesting epigenetic repression. Mechanistically, HIF-1α exhibited a phosphorylation-dependent band shift under hypoxia but not LPS. Although both conditions showed comparable overall phosphorylation levels by Phos-tag analysis, only hypoxia triggered a conformational change, suggesting site-specific phosphorylation linked to transcriptional competence. These findings demonstrate that HIF-1α binding alone is insufficient for gene activation and that phosphorylation and chromatin context determine its transcriptional output in a stimulus-dependent manner.

Abstract Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell–cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.