Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts

doi:10.1016/j.yjmcc.2022.05.012

科研成果详情

题名	Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts
作者	Nicks, Amy M.1,2; Holman, Sara R.1; Chan, Andrea Y.1; Tsang, Michael 1; Young, Paul E.1; Humphreys, David T.1,2; Naqvi, Nawazish 3; Husain, Ahsan 3; Li, Ming 1,2,5; Smith, Nicola J.1,2; Iismaa, Siiri E.1,2,4; Graham, Robert M.1,2,4
发表日期	2022-09
发表期刊	JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 影响因子和分区
语种	英语
原始文献类型	Article
关键词	Cardiomyocyte biology Cardiomyocyte isolation Postnatal heart development Cardiac hypertrophy Cardiomyocyte maturation Sex differences
其他关键词	GENE-EXPRESSION ; MOUSE ; FAILURE ; EPIDEMIOLOGY ; DISEASE ; GROWTH ; CELLS
摘要	Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2 x 106 cells/heart) and viability (-70-100%) of cardiomyocytes postisolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (-95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (-94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.
资助项目	National Health and Medical Research Council of Australia [573732, 1074386]; Heart Foundation of Australia [G10S 5148, 101153]; Australian Research Council Special Research Initiative in Stem Cell Science [SR110001002]; Leducq Transatlantic Network of Excellence in Cardiovascular Research
出版者	ELSEVIER SCI LTD
出版地	OXFORD
ISSN	0022-2828
EISSN	1095-8584
卷号	170 页码:47-59
DOI	10.1016/j.yjmcc.2022.05.012
页数	13
WOS类目	Cardiac & Cardiovascular Systems ; Cell Biology
WOS研究方向	Cardiovascular System & Cardiology ; Cell Biology
WOS记录号	WOS:000811351000001
收录类别	SCIE ; PUBMED
PubMed ID	35644482
通讯作者地址	[Iismaa, Siiri E.;Graham, Robert M.]Victor Chang Cardiac Res Inst, Darlinghurst, NSW 2010, Australia.;Graham, Robert M.]Univ NSW, St Vincents Clin Sch, Kensington, NSW 2052, Australia.;Graham, Robert M.]Victor Chang Cardiac Res Inst, 405 Liverpool St, Darlinghurst, NSW 2010, Australia.
引用统计	被引频次：1[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/148140
专题	基础医学院（机能实验教学中心）_基础医学研究中心_心脏再生医学研究所
通讯作者	Iismaa, Siiri E.; Graham, Robert M.
作者单位	1.Victor Chang Cardiac Res Inst, Darlinghurst, NSW 2010, Australia; 2.Univ NSW, St Vincents Clin Sch, Kensington, NSW 2052, Australia; 3.Emory Univ, Dept Med Cardiol, Sch Med, Atlanta, GA USA; 4.Victor Chang Cardiac Res Inst, 405 Liverpool St, Darlinghurst, NSW 2010, Australia; 5.Wenzhou Med Univ, Cardiac Regenerat Res Inst, Wenzhou 325035, Peoples R China
推荐引用方式 GB/T 7714	Nicks, Amy M.,Holman, Sara R.,Chan, Andrea Y.,et al. Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts[J]. JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY,2022,170:47-59.
APA	Nicks, Amy M.., Holman, Sara R.., Chan, Andrea Y.., Tsang, Michael., Young, Paul E.., ... & Graham, Robert M.. (2022). Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts. JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 170, 47-59.
MLA	Nicks, Amy M.,et al."Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts".JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 170(2022):47-59.