The Molecular Basis of Cardiac Senescence: From Transcriptomics to Function

心血管科学实验室致力于心肌老化的研究。为了鉴定心脏中可能与衰老有关的基因产物，功能基因组分析（cDNA微阵列和基因表达系列分析）已被用于分析来自有或没有热量限制的Fisher 344大鼠、Wistar大鼠、围产期和衰老的C57 B1/6小鼠、用涉及老年保护的生物肽处理的CBA小鼠、以及衰竭和非衰竭心肌的人体活检。这些项目的目的是确定哪些基因产物作为年龄或疾病的函数受到调节，然后使用独立的方法来确定导致基因表达改变的潜在机制。在2002年，我们发表了一个参考数据集（SAGE分析）的小鼠心肌是公开的，我们已经扩大了这些研究，以检查雄性与雌性小鼠和年轻与老年小鼠。 我们已经完成了一个大规模的转录组分析老化的Fisher 344老化大鼠模型，并比较这些结果与其他啮齿动物品系。我们正在完成一项广泛的定量PCR分析，以验证这些数据，并通过独立的技术，以确定基因表达的变化与衰老过程的意义。我们已经扩大了我们在啮齿类动物中的衰老研究，包括体外培养的心肌细胞和成纤维细胞的分析，以描绘心脏基因衰老反应的机制，并正在阐明这些转录本丰度变化的遗传相关性。 最后，我们采用功能基因组技术（微阵列）来检查LV的转录组从失败（n=8）和非失败的人心肌（n=7）。在通过微阵列和统计学方法鉴定了一组HF应答候选基因后，我们在较大的样本群体（n=34）上采用Q-PCR来验证和检查贡献生物变量（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）表现出基因表达的显着变化，然而，大多数假定的变化取决于变量，如性别和年龄，而不是单独的HF。此外，一些推定的HF反应基因产物表现出高度显着的变化，表达作为年龄和/或性别的函数，但独立于HF。 基于这些先前发表的数据和随后的微阵列分析使用预测分析微阵列软件，我们的研究工作集中在两个转录因子-Ets 1和Ets 2。它们在衰老中的具体作用尚未阐明，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之间的作用不同。Ets 2在心肌细胞中优先促凋亡，并且Ets 1和Ets 2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获得的组织样本，最近的数据表明，成纤维细胞与心肌细胞的凋亡机制不同，这取决于通过Ets 1和Ets 2的激活。

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 and recent data suggest that the mechanisms of apoptosis in fibroblasts versus cardiomyocytes differ depending on activation via Ets1 and Ets2.