Institute of Molecular Medicine, College of Life Science, Peking University, Beijing, China.
Circ Res. 2010 Feb 5;106(2):317-27. doi: 10.1161/CIRCRESAHA.109.208272. Epub 2009 Nov 19.
Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Ca(v)beta subunit of the L-type Ca(2+) channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation-contraction (EC) coupling is unknown.
This study aimed to investigate possible role of Rad in cardiac EC coupling and beta-adrenergic receptor (betaAR) inotropic mechanism.
Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (I(Ca)), Ca(2+) transients, and contractility by 60%, 42%, and 38%, respectively, whereas the "gain" function of EC coupling was significantly increased, due perhaps to reduced "redundancy" of LCC in triggering sarcoplasmic reticulum release. Conversely, approximately 70% Rad knockdown by RNA interference increased I(Ca) (50%), Ca(2+) transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on I(Ca). Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na(+)/Ca(2+) exchange activity, sarcoplasmic reticulum Ca(2+) content, properties of Ca(2+) sparks and propensity for Ca(2+) waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated betaAR effects on I(Ca) and Ca(2+) transients.
These results establish Rad as a novel endogenous regulator of cardiac EC coupling and betaAR signaling and support a parsimonious model in which Rad buffers Ca(v)beta to modulate LCC activity, EC coupling, and betaAR responsiveness.
Ras 相关糖尿病蛋白（Rad）是一种单体小分子 G 蛋白，与 L 型钙通道（LCC）的 Ca(v)beta 亚基结合，从而调节 LCC 的转运和活性。新出现的证据表明，Rad 是心脏心律失常和肥厚重塑的重要参与者。然而，Rad 是否以及如何参与兴奋-收缩（EC）偶联的调节尚不清楚。
本研究旨在探讨 Rad 在心脏 EC 偶联和β-肾上腺素能受体（βAR）变力机制中的可能作用。
在大鼠心肌细胞中，通过腺病毒过表达 Rad 使其增加 3 倍，分别使 LCC 电流（I(Ca)）、Ca(2+) 瞬变和收缩力降低 60%、42%和 38%，而 EC 偶联的“增益”功能显著增加，这可能是由于 LCC 在触发肌浆网释放方面的“冗余”减少。相反，通过 RNA 干扰使 Rad 下调约 70%，增加 I(Ca)（50%）、Ca(2+) 瞬变（52%）和收缩力（58%），而不改变 EC 偶联效率；显性负突变 RadS105N 对 I(Ca)也有类似的作用。Rad 的上调导致 LCC 激活的去极化偏移，并加速 LCC 的时程依赖性失活；然而，Rad 的下调并没有改变这些特性。Na(+)/Ca(2+) 交换活性、肌浆网 Ca(2+) 含量、Ca(2+) 火花的特性和 Ca(2+) 波的倾向都保持不变，无论 Rad 如何操作。Rad 的过表达而非下调，否定了βAR 对 I(Ca)和 Ca(2+) 瞬变的作用。
这些结果确立了 Rad 作为心脏 EC 偶联和βAR 信号的新型内源性调节剂，并支持一种简约的模型，即 Rad 缓冲 Ca(v)beta 以调节 LCC 活性、EC 偶联和βAR 反应性。
