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

• 批准号: 6932009
• 负责人: RENNOLDS S OSTROM
• 金额: $ 21.9万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932009
• 关键词: Adenoviridae; G protein; adenylate cyclase; adrenergic receptor; cardiac myocytes; cell biology; cyclic AMP; enzyme activity; extracellular matrix; fibroblasts; fibrosis; gene expression; gene therapy; genetic manipulation; heart cell; heart failure; heart function; hormone receptor; hormone regulation /control mechanism; isozymes; laboratory mouse; laboratory rat; nonhuman therapy evaluation; prostaglandin receptor; protein localization; protein structure function; receptor coupling; second messengers; tissue /cell culture

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）：本申请旨在检验以下假设：腺苷酸环化酶（AC，生成第二信使，cAMP，CAMP的酶）在心脏成纤维细胞中的表达可能会限制心脏病中心脏纤维化。 I和其他人发现，AC的表达水平限制了G蛋白偶联受体（GPCR）激动剂引起的最大cAMP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，在心脏成纤维细胞中增加cAMP的细胞水平的药物会减少细胞增殖和胶原蛋白合成（有助于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。该提案的目的将在心脏成纤维细胞的主要培养物中表征这些作用，其中三种不同的AC同工型过表达。然后，从细胞研究中获得的知识将扩展到心脏失败动物模型的研究，以确定AC表达是否会减弱心脏纤维化的发展，从而改善心脏功能。 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).在心肌细胞中，AC6过表达的这种选择性作用是由于betaar和AC在小窝中的共定位引起的 - 质膜的微素，它排除了前列腺素受体。因此，AC表达在心肌细胞中被高度分裂，受体和效应子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在小窝中没有高度分室。因此，至关重要的是表征心脏成纤维细胞中GPCR和AC的共定位以定义AC同工型过表达策略，从而最大程度地增强了内源激素的抗纤维化作用。具体目的将评估AC3，4和6的过表达对cAMP形成和细胞外基质的产生/降解的影响，确定内源激素受体与这些AC同工型的功能偶联和隔室的作用耦合和隔室，并靶向AC经过过表达对这种治疗的心脏成纤维的质量降低，并降低了对心脏的影响。