Department of Physiology, University of Kentucky College of Medicine, Lexington, KY.
J Am Heart Assoc. 2013 Dec 12;2(6):e000459. doi: 10.1161/JAHA.113.000459.
The small GTPase Rad is a negative regulator of voltage-dependent L-type calcium channel current (ICaL); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad-depletion causes positive inotropic effects without inducing cardiac hypertrophy.
Ventricular myocytes from adult Rad(-/-) mice were isolated and evaluated by patch-clamp recordings for I(Ca,L) and action potentials, Ca(2+) transients, and sarcomere shortening. Maximum I(CaL) is elevated in Rad(-/-) (maximal conductance 0.35 ± 0.04 picoSiemens/picoFarad (pS/pF) wild-type; 0.61 ± 0.14 pS/pF Rad(-/-)), decay kinetics are faster, and I(Ca,L) activates at lower voltages (activation midpoint -7.2 ± 0.6 wild-type; -11.7 ± 0.9 Rad(-/-)) mimicking effects of β-adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad(-/-) (F340/380: 1.03 diastolic and 0.35 twitch for wild-type; 1.47 diastolic and 0.736 twitch for Rad(-/-)) and sarcomere shortening is enhanced (4.31% wild-type; 14.13% Rad(-/-)) at lower pacing frequencies. Consequentially, frequency-dependence of Ca(2+) transients is less in Rad(-/-), and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, +dP/dt was elevated at baseline and developed pressure was relatively nonresponsive to acute β-adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin-dependent protein kinase-sensitive calcium release is observed. Remarkably, Rad(-/-) hearts did not show hypertrophic growth despite elevated levels of diastolic calcium.
This study demonstrates that the depletion of Rad GTPase is equivalent to sympathomimetic β-adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTPase is a novel potential therapeutic target for Ca(2+)-homeostasis-driven positive inotropic support of the heart.
小 GTP 酶 Rad 是电压依赖性 L 型钙通道电流（ICaL）的负调节剂；然而，Rad 缺失对心肌细胞功能的影响尚不清楚。本研究的目的是验证 Rad 耗竭会导致正性肌力作用而不引起心肌肥厚的假设。
从成年 Rad(-/-) 小鼠中分离心室肌细胞，并通过膜片钳记录评估 ICaL 和动作电位、Ca(2+)瞬变和肌节缩短。Rad(-/-)中的最大 ICaL 升高（最大电导 0.35 ± 0.04 皮西门子/皮法拉（pS/pF）野生型；0.61 ± 0.14 pS/pF Rad(-/-)），衰减动力学更快，ICaL 在更低的电压下激活（激活中点-7.2 ± 0.6 野生型；-11.7 ± 0.9 Rad(-/-)），模拟β-肾上腺素能受体刺激的作用。Rad(-/-)中的舒张和抽搐钙升高（F340/380：1.03 舒张和 0.35 抽搐野生型；1.47 舒张和 0.736 抽搐 Rad(-/-)），在较低的起搏频率下肌节缩短增强（4.31%野生型；14.13% Rad(-/-)）。因此，Rad(-/-)中 Ca(2+)瞬变的频率依赖性降低，并且松弛的频率依赖性也变钝。在分离的工作心脏中也获得了类似的结果；主要是，基础状态下 +dP/dt 升高，急性β-肾上腺素能受体刺激对发展压的反应相对不敏感。在单细胞中，在亚生理频率下，观察到非刺激钙调蛋白依赖性蛋白激酶敏感的钙释放。值得注意的是，尽管舒张钙水平升高，Rad(-/-)心脏仍未表现出肥厚生长。
本研究表明，Rad GTP 酶的耗竭相当于拟交感神经β-肾上腺素能受体，而不刺激心肌肥厚。因此，靶向 Rad GTP 酶是一种治疗 Ca(2+)稳态驱动的心脏正性肌力支持的新型潜在治疗靶点。
