Single Cell Spatial Transcriptomics Redefines the Borderzone induced by Myocardial Infarction and Mechanical Injury

The ischemic borderzone (BZ) is a geographically complex and biologically enigmatic interface separating poorly perfused infarct zones (IZ) from comparatively healthy remote zones (RZ). BZ cellular and molecular mechanisms are not well understood because efforts to dissect it inevitably include RZ and IZ in uncontrolled proportions. Here, we use single-cell/nuclei RNA-sequencing, spatial transcriptomics, and multiplexed RNA fluorescence in situ hybridization (mFISH) to identify BZ cardiomyocytes (CMs) subsets. BZ1 (Nppa+Xirp2­-) forms a hundreds-of-microns-thick transitional layer adjacent to RZ, while BZ2 (Nppa+Xirp2­+) forms a tens-of-microns-thick layer that the IZ edge. BZ2 CMs have reduced CM cell contact; colocalize with matricellular-protein-expressing myofibroblasts; and upregulate focal adhesion-, sarcomere-, and cytoskeletal-genes. Surprisingly, the transcriptional BZ emerges within an hour of injury and is inducible by non-ischemic fine-needle-trauma. We suggest that mechanical instability and “loss of neighbor” at the BZ edge are the dominant inducers of the BZ transcriptional response. Overall design: Gene expression profiling of 10x Genomic Chromium single nuclei RNA-Seq or Visium spatial transcriptomics from the left ventricular myocardium or short axis sections of healthy and injured adult male C57BL6 mouse hearts (8-12 weeks). Injuries include MI (permanent ligation of the LAD coronary artery), I/R-30 (30 minutes of ischemia followed by reperfusion), transaortic constriction, isoproterenol injection, and traumatic needle injection. Please note that the processed data files for GSM6613081, GSM6613087, GSM6613090 have been replaced on May 18, 2023 as the previous files were incomplete (missing spatial data folder and .h5 matrix file).

缺血边界区（ischemic borderzone, BZ）是一个空间结构复杂且生物学特性尚不明确的界面，将灌注不良的梗死区（infarct zone, IZ）与相对健康的远隔区（remote zone, RZ）分隔开来。由于任何旨在解析该区域的研究都不可避免地会混入比例未受控制的RZ与IZ样本，因此BZ的细胞与分子机制至今尚未被充分阐明。本研究采用单细胞/单细胞核RNA测序、空间转录组学以及多重RNA荧光原位杂交（multiplexed RNA fluorescence in situ hybridization, mFISH）技术，对BZ中心肌细胞（cardiomyocytes, CMs）的亚型进行鉴定。其中，BZ1亚型（Nppa⁺Xirp2⁻）形成一层厚度达数百微米的过渡层，紧邻RZ；而BZ2亚型（Nppa⁺Xirp2⁺）则形成一层仅数十微米厚的过渡层，毗邻IZ边缘。BZ2型心肌细胞的细胞间连接减少，与表达基质细胞蛋白的肌成纤维细胞共定位，并上调黏着斑、肌节及细胞骨架相关基因的表达。令人意外的是，转录层面的BZ特征可在损伤发生后1小时内出现，且可由非缺血性细针穿刺创伤诱导。我们推测，BZ边缘的机械稳定性缺失以及“邻近细胞丢失”是驱动BZ产生转录应答的主要诱因。研究总体设计：对8~12周龄健康及损伤后的成年雄性C57BL6小鼠心脏的左心室心肌组织或短轴切片，采用10x Genomic Chromium平台开展单细胞核RNA测序或Visium空间转录组学的基因表达谱分析。损伤模型包括心肌梗死（myocardial infarction, MI）（永久结扎左前降支冠状动脉）、I/R-30（30分钟缺血后再灌注）、主动脉缩窄术、异丙肾上腺素注射以及针穿刺创伤。请注意：由于此前的文件存在缺失（缺少空间数据文件夹及.h5矩阵文件），GSM6613081、GSM6613087、GSM6613090的处理后数据文件已于2023年5月18日完成替换。