背景:慢性交感神经刺激在心力衰竭中引起β1肾上腺素能受体(β1AR)的脱敏和下调。我们旨在探索心脏中β1AR信号传导的差异下调亚细胞池。
结果:我们在雄性C57BL/6J小鼠中应用了异丙肾上腺素的慢性输注来诱导心肌病。我们应用共聚焦和邻近连接测定法来检查β1AR与L型钙通道的相关性,ryanodine受体2和SERCA2a((Sarco)内质网钙ATPase2a)和基于Frster共振能量转移的生物传感器可探测心室肌细胞中的亚细胞β1AR-PKA(蛋白激酶A)信号。慢性输注异丙肾上腺素导致β1AR蛋白水平降低,通过邻近连接测量的与L型钙通道和ryanodine受体2的受体关联(puncta/cell,29.65生理盐水与14.17异丙肾上腺素,P<0.05),和受体诱导的质膜上的PKA信号(Förster共振能量转移,28.9%盐水与1.9%异丙肾上腺素,P<0.05)和ryanodine受体2复合物(Förster共振能量转移,30.2%生理盐水与10.6%异丙肾上腺素,P<0.05)。然而,β1AR与SERCA2a的关联增强(puncta/细胞,51.4盐水与87.5异丙肾上腺素,P<0.05),受体信号受到的影响最小。输注异丙肾上腺素的心脏显示PDE4D(磷酸二酯酶4D)和PDE3A减少,PDE2A增加,PDE4A,和PDE4B蛋白水平。我们观察到PDE4的作用降低,PDE2和PDE3对ryanodine受体2复合物和肌细胞缩短的β1AR-PKA活性的作用增强。尽管β1AR与SERCA2a的相关性增强,内源性去甲肾上腺素诱导的信号在SERCA2a复合物处减少。抑制单胺氧化酶A挽救了去甲肾上腺素诱导的SERCA2a处的PKA信号和肌细胞缩短。
结论:本研究揭示了慢性肾上腺素能刺激下心脏亚细胞β1AR信号下调的独特机制。
BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.
RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the β1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening.
CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular β1AR signaling in the heart under chronic adrenergic stimulation.