Caveolin-3 and cardiac fibrosis

项目总结/摘要 心血管疾病是65岁以上美国人死亡的主要原因。机制 心血管老化没有很好的定义，也没有有效的治疗方法。这个项目提出了一个新的 假设：年龄相关的心肌细胞表达小窝和蛋白小窝蛋白-3的损失， 由此产生的对心脏成纤维细胞的促纤维化活性的刺激，从而增强细胞外基质的产生， 基质、心脏纤维化、舒张功能障碍和心力衰竭，尤其是射血功能保留的心力衰竭 分数（HFpEF）。HFpEF是一种临床问题，目前尚无有效的治疗方法。这一假设源于 我们的初步数据显示，老年小鼠的心肌细胞中Caveolae表达缺失， 他们的关键常驻蛋白，小窝蛋白-3，从老年动物分离的心脏成纤维细胞， 纤维化活性，我们在离体培养的成纤维细胞中检测到。我们提出的研究将测试是否小窝蛋白-3在 心肌细胞通过减少心脏成纤维细胞的促纤维化状态而在衰老中具有心脏保护特性， 从而减缓与衰老相关的心脏纤维化的发展和进程。此外，我们将确定， 随着年龄的增长，小窝蛋白-3丢失的恢复可以减少年龄相关的心脏纤维化。我们的两个具体 目的会问：1）心肌细胞中caveolin-3的表达是否调节心脏的促纤维化状态 成纤维细胞和心脏纤维化和2）心肌细胞中小窝蛋白-3的恢复是否降低年龄- 与心脏纤维化有关吗在目标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个月）和老年（18-22个月）野生型、小窝蛋白-3-敲除和心肌细胞， 靶向小窝蛋白-3过表达小鼠。我们还将进行心肌细胞的RNA-seq研究， 心脏成纤维细胞，并将使用来自心肌细胞的条件培养基来评估外泌体和可溶性 年轻和年老的心脏释放到介质中的蛋白质。除了生理老化的研究，我们将 使小鼠经受经主动脉收缩，其诱导心脏纤维化，以便增加 纤维化改变在目标2中，我们将测试工程化以增加小窝蛋白-3表达的AAV构建体的影响。 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，建议的研究应该是高度可行的，甚至 尽管它们是R21应用中所寻求的高风险/高回报类型。我们认为， 将促进对衰老相关的心脏纤维化和功能障碍的理解， 方法-增加caveolin-3在心脏中的表达-是心脏病发病率和死亡率的主要贡献者。 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.