Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
Department of Medicine, School of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
Stem Cells. 2024 May 15;42(5):445-459. doi: 10.1093/stmcls/sxae016.
Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle."
Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle-specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5).
The cell-cycle-specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction.
The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs.
我们之前对来自胎儿猪和在出生后第 1 天（ARP1）接受心尖切除术手术、第 28 天（MIP28）接受心肌梗死（MI）手术、两者均接受 ARP1 和 MIP28（ARP1MIP28）或对照组（未进行手术或 CTL）的猪的心肌细胞单核 RNA 测序（snRNAseq）数据进行了分析，确定了 10 个心肌细胞亚群（簇），其中一个似乎对 MI 后的增殖有反应。然而，主要由增殖心肌细胞组成的簇仍然包含非循环细胞，并且我们无法区分处于细胞周期不同阶段的心肌细胞。在这里，我们通过仅对“细胞周期”GO 术语下包含的 1646 个基因进行 snRNAseq 数据分析，提高了我们评估的准确性。
评估了来自小鼠（GEO 数据集编号 GSE130699）和猪（GEO 数据集编号 GSE185289）的两个心脏 snRNAseq 数据集，通过我们的细胞周期特异性分析管道进行评估。通过共表达 5 个增殖标志物（AURKB、MKI67、INCENP、CDCA8 和 BIRC5）来鉴定有丝分裂细胞。
细胞周期特异性自动编码器（CSA）算法在小鼠心脏中鉴定出 7 个心肌细胞簇（mCM1 和 mCM3-mCM8），包括在 P1 接受 MI 或 Sham 手术的动物中一个突出的有丝分裂细胞簇。在猪心脏中鉴定出 5 个心肌细胞簇（pCM1、pCM3-pCM6），其中 2 个（pCM1 和 pCM4）显示出细胞周期活性的证据；pCM4 主要存在于胎儿猪的心脏中，而 pCM1 在 MI 诱导后 2 周的胎儿心脏和 ARP1MIP28 猪的心脏中构成了一小部分心肌细胞，但在所有其他实验组和所有其他时间点均无法检测到。此外，对来自胎儿猪心肌细胞的 snRNAseq 数据进行的伪时间轨迹分析确定了一条从 pCM3 开始、通过 pCM2 结束于 pCM1 的途径，而 pCM3 富含调节 G1/S 期转变的细胞周期激活物（cyclin D2），pCM2 富含 S 期调节剂（CCNE2），pCM1 富含调节 G2M 期转变和有丝分裂的基因（cyclin B2）的表达。我们还鉴定了 4 个转录因子（E2F8、FOXM1、GLI3 和 RAD51），它们在再生小鼠心脏中的心肌细胞中的表达比非再生小鼠心脏中的心肌细胞、比 CTL 猪心脏中的心肌细胞以及比 MI 诱导后 2 周的 MIP28 猪心脏中的心肌细胞中的表达更丰富。
CSA 算法提高了我们对心肌细胞亚群细胞周期活性评估的准确性，并使我们能够鉴定出一条跨越 3 个簇的轨迹，该轨迹似乎追踪了胎儿猪心肌细胞细胞周期活性的开始和进展。
