Max Planck Institute for Experimental Medicine, Goettingen, Germany.
Br J Pharmacol. 2011 Jan;162(2):544-56. doi: 10.1111/j.1476-5381.2010.01067.x.
Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms.
Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and Ca²(+) transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16.
Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC₅₀ : 19 nM) that were abolished by antisauvagine-30 (10 nM, n= 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF₂ receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying Ca²(+) transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca²(+)/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and Ca²(+) transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra Ca²(+) increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93.
Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF₂ receptors in a cAMP/PKA- and Ca²(+)/CaMKII-dependent manner. This enhancement was accompanied by Ca²(+)-dependent arrhythmogenic effects mediated by PKA and CaMKII.
孤啡肽 2 在心衰中有益，但其中的细胞机制尚不完全清楚。本研究旨在探讨孤啡肽 2 对小鼠心肌细胞的功能作用，并阐明其潜在的信号通路和机制。
在室温下，以 0.5 Hz 的频率对小鼠心室肌细胞进行场刺激。通过边缘检测和荧光显微镜系统分别测量心肌细胞的缩短分数和Ca²⁺瞬变。使用抗总磷蛋白（PLN）和丝氨酸-16 磷酸化 PLN 的抗体对心肌细胞提取物进行 Western blot 分析。
孤啡肽 2 呈现时间和浓度依赖性的正性变力和变时作用（EC₅₀：19 nM），这种作用可被 sauvagine-30（10 nM，n=6）特异性拮抗剂阻断，后者是促肾上腺皮质激素释放因子（CRF）CRF₂受体的拮抗剂。孤啡肽 2（100 nM）增加了基础Ca²⁺瞬变的幅度，并降低了衰减的时间常数。孤啡肽 2 还增加了丝氨酸-16 位的 PLN 磷酸化。蛋白激酶 A（PKA）抑制剂 H89（2 µM）或 KT5720（1 µM），以及 Ca²⁺/钙调蛋白依赖性蛋白激酶 II（CaMKII）抑制剂 KN93（1 µM）均抑制了孤啡肽 2 对缩短和Ca²⁺瞬变的作用。此外，孤啡肽 2 还引起心律失常事件，包括细胞缩短和舒张期Ca²⁺增加。在预先用 KT5720 或 KN93 处理的细胞中，孤啡肽 2 诱导的心律失常事件显著减少。
孤啡肽 2 通过激活心肌细胞中的 CRF₂受体，以 cAMP/PKA 和 Ca²⁺/CaMKII 依赖的方式增强了小鼠心室肌细胞的收缩力。这种增强伴随着由 PKA 和 CaMKII 介导的 Ca²⁺依赖性心律失常效应。
