Heads R J, Latchman D S, Yellon D M
Hatter Institute for Cardiovascular Studies, Department of Academic and Clinical Cardiology, University College Hospital, London, UK.
J Mol Cell Cardiol. 1994 Jun;26(6):695-9. doi: 10.1006/jmcc.1994.1084.
Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) beta-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i.(ABSTRACT TRUNCATED AT 250 WORDS)
热休克蛋白70（HSP70）已被证明在许多生物系统中热耐受的诱导过程中发挥着重要作用。在诸如热、缺氧、缺血、乙醇和重金属等多种应激反应下，HSP70合成的增加与对随后更严重应激的保护以及对不同应激的交叉耐受相关。在这方面，已表明哺乳动物心脏暴露于亚致死性热处理或缺血后，会伴随HSP70升高而对随后的心肌缺血产生保护作用。然而，直到最近，HSP70能单独赋予热耐受的直接证据还仅限于用HSP70基因转染成纤维细胞，尽管其他人的初步数据表明，用HSP70基因转染H9c2心肌细胞可使其耐受无底物的缺氧。因此，本研究的目的是直接测试一种保留心肌细胞某些特征（与非心肌细胞相对）的心肌细胞系，通过转染单个HSP70基因是否能免受致死性热应激。将大鼠心脏来源的H9c2细胞用一种质粒转染，该质粒由一个强的、异源（人）β-肌动蛋白启动子驱动人HSP70基因的高水平表达（APr-HS70），或者用一个不含HSP70基因的类似对照质粒（pAPr-1 neo）转染。在用新霉素类似物G418筛选后，分离出几个克隆，通过用针对HSP70i的特异性单克隆抗体进行蛋白质印迹分析确定，这些克隆要么不表达HSP70（对照：pAPr-1 neo衍生），要么稳定表达高水平的可诱导型HSP70异构体（HSP70i）（APr-HS70衍生）。（摘要截短于250字）
