Masahiro Satoh1, Seitaro Nomura2, Mutsuo Harada3, Toshihiro Yamaguchi3, Toshiyuki Ko2, Tomokazu Sumida3, Haruhiro Toko3, Atsuhiko T Naito3, Norifumi Takeda3, Takashige Tobita4, Takanori Fujita5, Masamichi Ito3, Kanna Fujita3, Masato Ishizuka3, Taro Kariya3, Hiroshi Akazawa3, Yoshio Kobayashi6, Hiroyuki Morita3, Eiki Takimoto3, Hiroyuki Aburatani7, Issei Komuro8. 1. Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan; Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. 2. Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 3. Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 4. Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; Department of Cardiology, Tokyo Women's Medical University, Tokyo, Japan. 5. Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. 6. Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan. 7. Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. Electronic address: haburata-tky@umin.ac.jp. 8. Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address: komuro-tky@umin.ac.jp.
Abstract
BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
Authors: Farhan Chaudhry; Jenna Isherwood; Tejeshwar Bawa; Dhruvil Patel; Katherine Gurdziel; David E Lanfear; Douglas M Ruden; Phillip D Levy Journal: Front Cardiovasc Med Date: 2019-12-10