Adenylyl cyclase as a regulator of cardiac fibroblasts

腺苷酸环化酶作为心脏成纤维细胞的调节剂

基本信息

批准号：
7095172
负责人：
RENNOLDS S OSTROM
金额：
$ 21.39万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-01 至 2008-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): This application is designed to test the hypothesis that increased expression of adenylyl cyclase (AC, the enzyme that generates the second messenger, cAMP) in cardiac fibroblasts can limit cardiac fibrosis in heart disease. I and others have found that the expression level of AC limits the maximal generation of cAMP elicited by G protein-coupled receptor (GPCR) agonists. Therefore, increased expression of this enzyme enhances the response to hormones that regulate AC activity. Preliminary data demonstrate that agents which increase cellular levels of cAMP in cardiac fibroblasts decrease cell proliferation and collagen synthesis (two key cellular functions that contribute to cardiac fibrosis). Moreover, gene transfer of AC enhances this inhibition. The Aims of this proposal will characterize these effects in primary cultures of cardiac fibroblasts in which three different AC isoforms are overexpressed. The knowledge gained from cellular studies will then be extended to studies of an animal model of heart failure to determine if increased AC expression can attenuate the development of cardiac fibrosis and thereby improve cardiac function. However, my recent studies of cardiac myocytes and fibroblasts indicate that overexpression of a particular isoform of AC, AC6, increases the maximal cAMP generated by activation of beta-adrenergic receptors (betaAR) but does not enhance basal levels of cAMP or that stimulated by agents which activate other GPCR coupled to Gs (e.g. prostanoid receptors). In cardiac myocytes, this selective effect of AC6 overexpression is due to co-localization of betaAR and AC in caveolae - a microdomaln of the plasma membrane that excludes prostanoid receptors. Therefore, AC expression is highly compartmentalized in cardiac myocytes and co-localization of receptor and effector is essential for efficient signal transduction. In contrast, other studies I have conducted in vascular smooth muscle cells indicate that betaAR and AC are not highly compartmentalized in caveolae. Therefore, it is critical to characterize the co-localization of GPCR and AC in cardiac fibroblasts in order to define an AC isoform overexpression strategy that most efficaciously enhances the anti-fibrotic effect of endogenous hormones. The specific aims will assess the impact of overexpression of AC3, 4 and 6 on cAMP formation and extracellular matrix production/degradation, determine the functional coupling and compartmentation of endogenous hormonal receptors with these AC isoforms, and target AC transgene overexpression to cardiac fibroblasts of mice and assess if this treatment decreases cardiac fibrosis in an experimental model of heart failure.

描述（由申请人提供）：本申请旨在测试以下假设：增加心脏成纤维细胞中腺苷酸环化酶（AC，产生第二信使 cAMP 的酶）的表达可以限制心脏病中的心脏纤维化。我和其他人发现 AC 的表达水平限制了 G 蛋白偶联受体 (GPCR) 激动剂引发的 cAMP 的最大生成量。因此，这种酶的表达增加会增强对调节 AC 活性的激素的反应。初步数据表明，增加心脏成纤维细胞中 cAMP 细胞水平的药物会减少细胞增殖和胶原合成（导致心脏纤维化的两个关键细胞功能）。此外，AC 的基因转移增强了这种抑制作用。该提案的目的将描述心脏成纤维细胞原代培养物中的这些效应，其中三种不同的 AC 亚型被过表达。从细胞研究中获得的知识将扩展到心力衰竭动物模型的研究中，以确定AC表达的增加是否可以减弱心脏纤维化的发展，从而改善心脏功能。然而，我最近对心肌细胞和成纤维细胞的研究表明，AC、AC6 的特定亚型的过度表达会增加由 β-肾上腺素能受体 (betaAR) 激活产生的最大 cAMP，但不会提高 cAMP 的基础水平或药物刺激的水平它激活与 Gs 偶联的其他 GPCR（例如前列腺素受体）。在心肌细胞中，AC6 过表达的这种选择性效应是由于 betaAR 和 AC 在细胞膜窝（一种排除前列腺素受体的质膜微区）中共定位所致。因此，AC 表达在心肌细胞中高度区分开，受体和效应器的共定位对于有效的信号转导至关重要。相比之下，我在血管平滑肌细胞中进行的其他研究表明，βAR 和 AC 在小窝中并未高度区分开。因此，表征 GPCR 和 AC 在心脏成纤维细胞中的共定位至关重要，以便定义最有效增强内源激素抗纤维化作用的 AC 亚型过表达策略。具体目标将评估 AC3、4 和 6 的过表达对 cAMP 形成和细胞外基质产生/降解的影响，确定内源激素受体与这些 AC 亚型的功能耦合和区划，并将 AC 转基因过表达靶向小鼠的心脏成纤维细胞并评估这种治疗是否可以在心力衰竭实验模型中减少心脏纤维化。