Adenylyl cyclase as a regulator of cardiac fibroblasts

描述（由申请人提供）：本申请旨在检验心脏成纤维细胞中腺苷酸环化酶（AC，产生第二信使cAMP的酶）表达增加可限制心脏疾病中心脏纤维化的假设。我和其他人已经发现AC的表达水平限制了由G蛋白偶联受体（GPCR）激动剂引起的cAMP的最大产生。因此，这种酶的表达增加增强了对调节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的细胞水平的试剂降低细胞增殖和胶原蛋白合成（导致心脏纤维化的两种关键细胞功能）。此外，AC的基因转移增强了这种抑制作用。本提案的目的是描述三种不同AC亚型过表达的心脏成纤维细胞原代培养物中的这些效应。然后将从细胞研究中获得的知识扩展到心力衰竭动物模型的研究，以确定AC表达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的一种特定亚型AC 6的过表达增加了由β-肾上腺素能受体（β AR）激活产生的最大cAMP，但不增加cAMP的基础水平或由激活与Gs偶联的其他GPCR的试剂（例如前列腺素受体）刺激的cAMP水平。在心肌细胞中，AC 6过表达的这种选择性作用是由于β AR和AC在小窝中的共定位-小窝是质膜的微结构域，不包括前列腺素受体。因此，AC表达在心肌细胞中是高度区室化的，并且受体和效应子的共定位对于有效的信号转导是必不可少的。相反，我在血管平滑肌细胞中进行的其他研究表明，β AR和AC在小窝中没有高度区室化。因此，关键是表征心脏成纤维细胞中GPCR和AC的共定位，以定义最有效地增强内源性激素的抗纤维化作用的AC同种型过表达策略。具体目标将评估AC 3、4和6的过表达对cAMP形成和细胞外基质产生/降解的影响，确定内源性激素受体与这些AC亚型的功能偶联和区室化，并将AC转基因过表达靶向小鼠的心脏成纤维细胞，并评估该治疗是否减少心力衰竭实验模型中的心脏纤维化。

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.