Adenylyl cyclase as a regulator of cardiac fibroblasts

描述(申请人提供)：本申请旨在测试心脏成纤维细胞中腺酰环化酶(AC，产生第二信使cAMP的酶)的表达增加可以限制心脏病心脏纤维化的假说。我和其他人发现，AC的表达水平限制了G蛋白偶联受体(GPCR)激动剂诱导的cAMP的最大生成。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内cAMP水平的药物会降低细胞增殖和胶原合成(促成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。这项提案的目的将在心脏成纤维细胞的原代培养中表征这些影响，在这些细胞中，三种不同的AC亚型过表达。从细胞学研究中获得的知识随后将扩展到心力衰竭动物模型的研究中，以确定AC表达增加是否可以减缓心脏纤维化的发展，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AC6的过表达增加了由β-肾上腺素能受体(BetaAR)激活产生的最大cAMP，但不增加cAMP的基础水平或激活其他与Gs偶联的GPC的药物(如前列腺素受体)刺激的cAMP水平。在心肌细胞中，AC6过度表达的这种选择性效应是由于BetaAR和AC在小窝中的共同定位-小窝是质膜上排除前列腺素受体的微域。因此，AC在心肌细胞中的表达是高度区域化的，受体和效应器的共同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上进行的其他研究表明，在小窝中，β-AR和AC并不是高度分隔的。因此，为了确定一种最有效地增强内源性激素的抗纤维化作用的AC亚型过表达策略，确定GPCR和AC在心脏成纤维细胞中的共定位是至关重要的。具体目的将评估AC3、4和6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隔，并将AC转基因过表达靶向小鼠心脏成纤维细胞，并评估该疗法是否能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.