Single-cell transcriptomics reveal distinctive patterns of fibroblast activation in heart failure with preserved ejection fraction

Inflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease specific traits during adverse remodeling, we profiled interstitial cells in early murine HFpEF using single-cell RNAseq (scRNAseq). Diastolic dysfunction and perivascular fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Integration of fibroblasts from HFpEF with two murine models for heart failure with reduced ejection fraction (HFrEF) identified a catalog of conserved fibroblast phenotypes across mouse models. Moreover, HFpEF specific characteristics included induced metabolic, hypoxic and inflammatory transcription factors and pathways, including enhanced expression of Angiopoietin-like 4 (Angptl4) next to basement membrane compounds, such as collagen IV (Col4a1). Fibroblast activation was further dissected into transcriptional and compositional shifts and thereby highly responsive cell states for each HF model were identified. In contrast to HFrEF, where myofibroblast and matrifibrocyte activation were crucial features, we found that these cell states played a subsidiary role in early HFpEF. These disease-specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes. Furthermore, we identified a potential cross-talk between macrophages and fibroblasts via SPP1 and TNF? with estimated fibroblast target genes including Col4a1 and Angptl4. Treatment with recombinant ANGPTL4 ameliorated the murine HFpEF phenotype and diastolic dysfunction by reducing collagen IV deposition from fibroblasts in vivo and in vitro. In line, ANGPTL4, was elevated in plasma samples of HFpEF patients and particularly high levels associated with a preserved global longitudinal strain. Taken together, our study provides a comprehensive characterization of molecular fibroblast activation patterns in murine HFpEF, as well as the identification of Angiopoietin-like 4 as central mechanistic regulator with protective effects. Overall design: Comparing effects of HFpEF treamtent (L-NAME +HFD) mouse model with control. We sequenced ventricular tissue of 2x control and 2x HFpEF mice.

炎症、纤维化与代谢应激是射血分数保留型心力衰竭（heart failure with preserved ejection fraction, HFpEF）发生发展的关键促发因素。高脂饮食（high-fat diet, HFD）与一氧化氮合酶抑制剂N[w]-硝基-L-精氨酸甲酯（N[w]-nitro-l-arginine methyl ester, L-NAME）暴露可在小鼠体内重现射血分数保留型心力衰竭的病理特征。为明确不良心肌重构过程中的疾病特异性特征，本研究采用单细胞RNA测序（single-cell RNAseq, scRNAseq）对早期小鼠射血分数保留型心力衰竭模型的间质细胞开展转录组图谱分析。

舒张功能障碍与血管周围纤维化伴随心脏成纤维细胞与巨噬细胞亚群的激活。将射血分数保留型心力衰竭模型小鼠的成纤维细胞与两种射血分数降低型心力衰竭（heart failure with reduced ejection fraction, HFrEF）小鼠模型的成纤维细胞进行整合分析，鉴定出跨小鼠心力衰竭模型的保守成纤维细胞表型谱。此外，射血分数保留型心力衰竭的特异性特征包括代谢、缺氧与炎症相关转录因子及通路的激活，除基底膜成分（如IV型胶原（collagen IV, Col4a1））外，还伴随血管生成素样4（Angiopoietin-like 4, Angptl4）表达上调。

研究进一步将成纤维细胞激活拆解为转录组与细胞组成的动态变化，从而鉴定出每种心力衰竭模型对应的高响应细胞状态。与射血分数降低型心力衰竭以肌成纤维细胞与基质成纤维细胞激活为核心特征不同，我们发现此类细胞状态在早期射血分数保留型心力衰竭中仅发挥次要作用。上述疾病特异性成纤维细胞特征在人类心肌批量转录组（bulk transcriptomes）中得到了验证。

此外，本研究还鉴定出巨噬细胞与成纤维细胞之间可能通过SPP1与肿瘤坏死因子α（tumor necrosis factor α, TNF-α）实现跨细胞通讯，预测的成纤维细胞靶基因包括Col4a1与Angptl4。体内外实验证实，重组ANGPTL4可通过减少成纤维细胞分泌的IV型胶原沉积，改善小鼠射血分数保留型心力衰竭表型与舒张功能障碍。与此一致，射血分数保留型心力衰竭患者血浆样本中ANGPTL4水平升高，且高表达水平与保留的整体纵向应变（global longitudinal strain, GLS）相关。

综上，本研究全面解析了小鼠射血分数保留型心力衰竭中成纤维细胞激活的分子模式，并鉴定出血管生成素样4作为核心机械调控因子，其可发挥保护作用。

实验设计：对比射血分数保留型心力衰竭造模组（L-NAME联合高脂饮食）小鼠与对照组的差异。本研究对2只对照组小鼠与2只射血分数保留型心力衰竭模型小鼠的心室组织进行了转录组测序。