Contractile Regulation of Cardiocyte Protein Synthesis

描述(由申请人提供)： 部分或完全闭塞冠状动脉会造成缺血，从而使心肌暴露在供氧不足(缺氧)或完全缺氧(缺氧)的环境中。因此，心肌损伤和坏死的发生依赖于多个变量，如缺氧的程度和持续时间，以及心肌细胞在心肌复氧(缺血/再灌注)过程中产生的不良影响。在正常生理条件下，心肌细胞的整体能量能力与脂肪酸氧化代谢燃料和线粒体氧化磷酸化产生三磷酸腺苷有关。为了最大限度地减少缺氧对心脏结构和功能的不利影响，心肌通过激活糖酵解途径来调节心肌细胞的能量代谢。这种向葡萄糖代谢的转变是在缺血性心脏病和其他心力衰竭原因期间发生的病理重塑的共同特征。这种能量代谢的基因程序受雌激素相关受体(ERR)的调控，ERR是核受体超家族的成员，它激活线粒体生物发生、脂肪酸代谢和氧化磷酸化所需的一系列靶基因的转录。具体地说，这些研究将通过检测调节ERR mRNA亚型翻译效率和/或稳定性的分子机制来确定导致ERR靶基因下调的原因，以响应成年心肌细胞的缺氧和复氧。ERR mRNA在54-UTR和34-UTR中既有结构特征，又有序列元件，这是翻译调控mRNAs的特征。其中包括一个富含G-C的54-UTR(82%)，它在54-帽附近以大而稳定的发夹环的形式存在着广泛的二级结构，以及一个相对较长的34-UTR，它可能含有富含AU的元件，参与了翻译相关的mRNA稳定性。初步研究表明，在缺氧和复氧过程中，成年心肌细胞ERR1的表达在翻译水平上受到调控。这些研究将考察低氧和复氧在调节成年猫心肌细胞ERR mRNA异构体翻译效率和稳定性方面的影响，成年猫心肌细胞在电刺激下以特定的生理参数持续收缩。将使用小鼠心肌缺血和缺血/再灌注损伤模型进行配套研究，以确定成人心肌细胞模型对体内氧化应激的生理学意义。假设在成年心肌细胞缺氧和复氧过程中，ERR1的表达是通过改变ERR1mRNA的翻译和/或稳定性的机制来控制的。其具体目的是：1)确定在体外培养的成年心肌细胞缺氧和缺氧/复氧过程中，以及在小鼠缺血再灌注损伤后的心肌重塑过程中，调节ERR基因表达的机制(S)；2)研究54-UTR和/或34-UTR中的特定元件在调节成年心肌细胞ERR1mRNA的翻译和稳定性中的作用；3)检测起始因子的修饰活性如何改变ERR1mRNA的表达及其活性(通过下游对靶基因的影响来确定)。这些研究的长期目标是更好地了解在不利重塑过程中导致心肌基因表达适应性变化的机制，并最终设计出能够逆转向心力衰竭过渡并改善美国退伍军人和其他患者预后的治疗策略。

DESCRIPTION (provided by applicant): Partial or complete occlusion of a coronary artery creates ischemia, thereby exposing the myocardium to either an insufficient supply of oxygen (hypoxia) or a complete lack of oxygen (anoxia). Consequently, myocardial injury and necrosis occur that are dependent on multiple variables such as the extent and duration of oxygen deprivation and the adverse effects produced in cardiomyocytes during reoxygenation of the myocardium (ischemia/reperfusion). The overall energetic capacity of cardiomyocytes under normal physiological conditions is coupled to fatty acid oxidation for metabolic fuel and to mitochondrial oxidative phosphorylation for ATP generation. To minimize the adverse effects of insufficient oxygen on cardiac structure and function, the myocardium adapts by activating glycolytic pathways to fuel energy metabolism in the cardiomyocyte. This switch to glucose metabolism is a common feature of pathological remodeling that develops during ischemic heart disease and other causes of heart failure. This gene program for energy metabolism is regulated by Estrogen-Related Receptors (ERR), a member of the nuclear receptor superfamily that activates transcription of a wide array of target genes required for mitochondrial biogenesis, fatty acid metabolism and oxidative phosphorylation. Specifically, these studies will define the causes that result in down-regulation of ERR target genes by examining molecular mechanisms that regulate translational efficiency and/or stability of ERR mRNA isoforms in response to hypoxia and reoxygenation of adult cardiocytes. ERR mRNA has both structural features and sequence elements in the 54-UTR and 34-UTR that are characteristic of translationally regulated mRNAs. These include a 54-UTR that is G-C rich (82%) with extensive secondary structure in the form of a large, stable hairpin loop near the 54-cap, and a relatively long 34-UTR that has possible AU-rich elements involved in translation-linked mRNA stability. The preliminary data have revealed that ERR1 expression is regulated in adult cardiomyocytes during hypoxia and reoxygenation at the level of translation. These studies will examine the effects of hypoxia and reoxygenation in regulating translational efficiency and stability of ERR mRNA isoforms in adult feline cardiomyocytes that are electrically stimulated to contract continuously at defined physiological parameters. Companion studies will be done using a murine model of myocardial ischemia and ischemia/reperfusion injury establish the physiological significance of the adult cardiomyocyte model to oxidative stress in vivo. The hypothesis is that expression of ERR1 is controlled during hypoxia and reoxygenation in the adult cardiomyocyte by mechanisms that modify translation and/or stability of ERR1 mRNA. The specific aims are: 1) to determine the mechanism(s) that regulate expression of ERRs during hypoxia and hypoxia/reoxygenation of adult cardiomyocytes in vitro and during myocardial remodeling produced in response to ischemia and ischemia/reperfusion injury in a murine model in vivo; 2) to examine the role of specific elements in the 54-UTR and/or 34-UTR in regulating translation and stability of ERR1 mRNA in adult cardiomyocytes; 3) to examine how modifying activity of initiation factors can alter expression of ERR1 and its activity as determined by downstream effects on target genes. The long term goal of these studies to gain a better understanding of the mechanisms that contribute to adaptive changes in myocardial gene expression during adverse remodeling, and ultimately devise therapeutic strategies that can reverse the transition to heart failure and improve outcomes for Veterans and other patients in the U.S. population.