The Molecular Basis of Cardiac Senescence: From Transcriptomics to Function

心血管科学实验室致力于老化心肌的研究。为了确定心脏中可能与衰老有关的基因产物，我们采用了功能基因组分析(基因芯片和基因表达系列分析)来分析Fisher 344大鼠(无论是否有热量限制)、Wistar大鼠、C57BL/6小鼠在围产期和衰老期间的左心室mRNA、接受老年保护作用的生物肽治疗的CBA小鼠以及衰竭和非衰竭心肌的人活组织。这些项目的目的是确定哪些基因产物作为年龄或疾病的功能受到调节，然后使用独立的方法来确定导致基因表达变化的潜在机制。2002年，我们发布了公开的小鼠心肌参考数据集(SAGE分析)，我们扩大了这些研究的范围，以检查雄性小鼠与雌性小鼠以及年轻小鼠与老年小鼠。 我们已经完成了对Fisher 344衰老大鼠模型的大规模转录组分析，并将这些结果与其他啮齿动物品系进行了比较。我们正在完成一项广泛的定量-聚合酶链式反应分析，以验证这些数据，并通过独立的技术来确定基因表达随衰老过程的变化的意义。我们已经扩展了我们对啮齿动物的衰老研究，包括对培养的心肌细胞和成纤维细胞的体外分析，以描绘心脏基因衰老反应的潜在机制，并正在阐明这些转录丰度变化背后的遗传相关性。 最后，我们使用功能基因组技术(微阵列)检测了衰竭(n=8)和非衰竭(n=7)人心肌LV的转录。在通过微阵列和统计方法确定了一组对HF有反应的候选基因库之后，我们在更大的样本人群(n=34)上使用了Q-PCR来验证和检验生物变量(年龄和性别)的作用。我们发现，大多数HF候选基因(包括转录因子、修饰酶、ECM蛋白和代谢酶)都显示出显著的基因表达变化；然而，大多数假定的变化取决于性别和年龄等变量，而不是仅仅依赖于HF。此外，一些假定的HF反应基因产物显示，随着年龄和/或性别的变化，表达发生了非常显著的变化，但与HF无关。 基于这些先前发表的数据和随后使用微阵列预测分析软件进行的微阵列分析，我们的研究工作集中在两个转录因子-Ets1和Ets2上。它们在衰老中的具体作用尚不清楚，但从体外分析来看，这些因子是促凋亡的；然而，成纤维细胞和心肌细胞的作用不同。Ets2在心肌细胞中优先促进凋亡，并且Ets1和Ets2都可以被血管紧张素II上调。这些发现现在正在扩展到从老年啮齿动物身上获得的组织样本，数据正在准备发表。

The Laboratory of Cardiovascular Science has a strong commitment to the study of aging myocardium. To identify gene products in heart potentially involved in aging, functional genomic analyses (cDNA microarrays and Serial Analysis of Gene Expression) have been employed to analyze mRNA from left ventricles of Fisher 344 rats with or without caloric restriction, Wistar rats, C57Bl/6 mice during the perinatal period and with aging, CBA mice treated with biopeptides implicated in gerontoprotection, and human biopsies from failing and non-failing myocardium. The aim of these projects are to determine which gene products are regulated as a function of age or disease, and then use independent methods to determine the underlying mechanisms responsible for the altered changes in gene expression. In 2002, we published a reference dataset (SAGE analysis) of the mouse myocardium that is publicly available, and we have expanded these studies to examine male versus female mice and young versus old mice. We have completed a large-scale transcriptome analysis of an aging Fisher 344 aging rat model and compared these results with other rodent strains. We are completing an extensive quantitative-PCR analysis to validate these data, and by independent techniques, to determine the significance of the changes in gene expression with the aging process. We have expanded our aging studies in rodent to include in vitro analyses of cultured cardiomyoyctes and fibroblasts to delineate mechanisms underlying the aging response of cardiac genes, and are in the process of elucidating genetic correlates underlying these changes in transcript abundance. Finally, we have employed functional genomic techniques (microarrays) to examine the transcriptomes of LVs from failing (n=8) and non-failing human myocardium (n=7). Following identification of a pool of HF-responsive candidate genes by microarrays and statistical methods, we employed Q-PCR on a larger sample population (n=34) to validate and examine the role of contributing biological variables (age and gender). We find that most of the HF-candidate genes (including transcription factors, modifying enzymes, ECM proteins and metabolic enzymes) demonstrated significant changes in gene expression; however, the majority of the putative changes depended on variables such as sex and age, and not on HF alone. Additionally, some putative HF-responsive gene products demonstrated highly significant changes in expression as a function of age and/or sex, but independent of HF. Based on these previously published data and subsequent microarray analyses using Prediction Analysis of Microarray software, our research efforts have focused on two transcription factors - Ets1 and Ets2. Their specific roles in aging are yet to be elucidated, but from in vitro analyses, these factors are pro-apoptotic; however, the effects differ between fibroblasts and cardiomyocytes. Ets2 is preferentially pro-apoptotic in cardiomyocytes, and both Ets1 and Ets2 can be up-regulated by angiotensin II. These findings are now being expanded to tissue samples obtained from aged rodents and the data are being prepared for publication.