砂河 孝行

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.

Cancer-stromal interactions play important roles in the biology of various cancers, including lung adenocarcinoma. We aimed to comprehensively analyze the lung cancer interactome and identify the key ligand-receptor pairs involved in the aggressiveness of lung adenocarcinoma. Transcriptome data were obtained from xenografts of 11 lung cancer cell lines that represented the major driver mutations in lung adenocarcinomas. A quantitative dataset was constructed in both stroma-to-cancer and cancer-to-stroma directions using the cancer-stromal interactome analysis method. The prognostic value of each factor was evaluated using multiple datasets. Analysis of 24,250 stroma-derived mouse transcripts and 26,289 human cancer-derived transcripts identified 1150 cancer-stromal interactions, from which we selected 117 interactions based on the intensity score of ligand-stromal transcript levels. Further prognostic analysis using public databases led us to identify 21 ligand-receptor pairs, including well-known as well as less well-characterized ligand-receptor pairs. Therefore, we selected tumor necrosis factor superfamily member 12/tumor necrosis factor receptor superfamily member 12A as possible factors contributing to the aggressiveness of lung adenocarcinoma via cancer-stromal interactions; immunohistochemical analysis confirmed that these factors were expressed mainly in the stroma and cancer cells, respectively, in both xenografts and primary lung adenocarcinoma. In human clinical specimens, high tumor necrosis factor receptor superfamily member 12A expression significantly correlated with tumor size, invasive diameter, and stage. Thus, tumor necrosis factor superfamily member 12 and its receptor tumor necrosis factor receptor superfamily member 12A signaling axis may be potential candidates for therapeutic intervention for lung adenocarcinoma.

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 ChIP-seq 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, while 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 non-induced 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.