Caveolin-3 and cardiac fibrosis

项目摘要/摘要 心血管疾病是65岁以上美国人的主要死亡原因。机制： 心血管老化没有明确的定义，也没有有效的治疗方法。这个项目提出了一项新的 假设：与年龄相关的小凹心肌细胞表达缺失和小窝蛋白-3的表达 由此刺激心脏成纤维细胞的促纤维化活性，从而增加细胞外物质的产生 基质、心脏纤维化、舒张期功能障碍和心力衰竭，尤其是射血功能不全的心力衰竭 分数(HFpEF)。HFpEF是一个临床问题，目前还没有有效的治疗方法。这一假设源自于 我们的初步数据表明，老年小鼠心肌细胞有小凹和小窝的表达缺失 他们的关键驻留蛋白小窝蛋白-3和从老年动物中分离的心脏成纤维细胞增加了对蛋白质的促进作用。 纤维化活性，我们在体外培养的成纤维细胞中检测到这一活性。我们提议的研究将测试小窝蛋白-3是否在 心肌细胞在衰老过程中通过减少心脏成纤维细胞的纤维化状态而具有心脏保护特性， 从而钝化衰老相关的心脏纤维化的发展和进展。此外，我们将确定是否 随着年龄的增加，修复小窝蛋白-3的丢失可以减少与年龄相关的心肌纤维化。我们的两个特别的 AIMS将问：1)心肌细胞中小窝蛋白-3的表达是否调节心脏的促纤维化状态 成纤维细胞与衰老中的心脏纤维化2)心肌细胞小窝蛋白-3的修复是否能降低年龄-- 相关的心脏纤维化？在目标1中，我们将评估心脏功能和心脏成纤维细胞的纤维化活性。 分离自年轻(2-3月)和老年(18-22月)野生型、小窝蛋白-3基因敲除和心肌细胞- 靶向小窝蛋白-3过表达的小鼠。我们还将对心肌细胞进行rna-seq研究，并 心脏成纤维细胞，并将使用心肌细胞的条件培养液来评估外切体和可溶性 蛋白质由年轻的和年老的心脏释放到介质中。除了对生理衰老的研究，我们还将 使小鼠经腹主动脉缩窄，诱发心肌纤维化，从而增加心脏收缩的动态范围。 纤维性改变。在目标2中，我们将测试AAV构建物对增加小窝蛋白-3表达的影响 在心肌细胞中对心功能、心肌细胞和心脏成纤维细胞的影响 目标1.根据我们的初步数据和过去的努力，拟议的研究甚至应该是高度可行的 尽管他们属于R21申请中寻求的高风险/高回报类型。我们相信，结果是 将促进对衰老相关的心脏纤维化和功能障碍的理解，并可能确定一种治疗方法 方法-增加心脏中小窝蛋白-3的表达-是导致心脏病发病率和死亡率的主要因素 老年人口。

Project Summary/Abstract Cardiovascular disease is the major cause of death in Americans over the age of 65. Mechanisms of cardiovascular aging are not well defined nor are effective treatments available. This project proposes a novel hypothesis: age-related loss in the expression by cardiac myocytes of caveolae and the protein caveolin-3 with the resultant stimulation of pro-fibrotic activity of cardiac fibroblasts, thereby enhancing production of extracellular matrix, cardiac fibrosis, diastolic dysfunction, and heart failure, especially heart failure with preserved ejection fraction (HFpEF). HFpEF is a clinical problem for which no effective therapies exist. This hypothesis derives from our preliminary data which show that cardiac myocytes of aged mice have a loss in expression of caveolae and their key resident protein, caveolin-3 and that cardiac fibroblasts isolated from aged animals have increased pro- fibrotic activity, which we detect in fibroblasts cultured ex vivo. Our proposed studies will test if caveolin-3 in cardiac myocytes has cardioprotective properties in aging by reducing the profibrotic state of cardiac fibroblasts, thus blunting the development and progression of aging-related cardiac fibrosis. In addition, we will determine if restoration of the loss in caveolin-3 with advanced age can reduce age-related cardiac fibrosis. Our two specific aims will ask: 1) Does caveolin-3 expression in cardiac myocytes regulate the pro-fibrotic state of cardiac fibroblasts and cardiac fibrosis in aging? and 2) Does restoration of caveolin-3 in cardiac myocytes reduce age- related cardiac fibrosis? In Aim 1, we will assess cardiac function and the fibrotic activity of cardiac fibroblasts isolated from young (2-3 month) and aged (18-22 month) wild-type, caveolin-3- knockout, and cardiac myocyte- targeted caveolin-3 over-expressing mice. We will also conduct RNA-seq studies of cardiac myocytes and cardiac fibroblasts and will use conditioned media from cardiac myocytes to assess exosomes and soluble proteins released into the media by young and old hearts. In addition to studies of physiological aging, we will subject mice to transaortic constriction, which induces cardiac fibrosis, so as to increase the dynamic range of fibrotic changes. In Aim 2, we will test the impact of AAV constructs engineered to increase caveolin-3 expression in cardiac myocytes on cardiac function, cardiac myocytes and cardiac fibroblasts using the approaches from Aim 1. Based on our preliminary data and past efforts, the proposed studies should be highly feasible even though they are of the high risk/high reward type that is sought in R21 applications. We believe that the results will advance understanding of aging-related cardiac fibrosis and dysfunction and may identify a therapeutic approach—increasing caveolin-3 expression in the heart—for a major contributor to morbidity and mortality in the aged population.