Tyrosine kinase receptors (VEGFR and EGFR) regulate normal automaticity of heart pacemaker, the sinoatrial node.

（一） 我们发现EGFR和VEGFR 1/2及其下游靶点PLC和PKC（通过RT-qPCR评估）在SA结中表达，其表达水平与心室中的表达水平相当。 （二） 特异性EGFR抑制剂AG 1478显著降低自发SANC搏动率（穿孔膜片钳技术），这种作用在药物洗脱后基本可逆，表明EGFR在基础状态下具有活性。在研究EGFR抑制对Ca 2+时钟的影响后，进一步阐明了AG 1478对自发SANC放电的影响。AG 1478显著抑制SANC中SR Ca 2+循环（共聚焦显微镜，Ca 2+指示剂Fluo-3AM），即减少每个自发循环的LCR大小和数量，并延长LCR周期（AP诱导的Ca 2+瞬变和随后的LCR之间的间隔），这预测了自发循环长度的伴随增加。我们还研究了VEGFR 1/2/3抑制剂vatalanib对SANC中LCR特征的影响。Vatalanib显著抑制LCR参数，即，减少每个自发循环的LCR大小和数量，延长LCR周期，并伴有自发循环长度的增加。Vatalanib还产生SANC的药物行为。总体而言，vatalanib使自发SANC搏动率降低了50%。然而，选择性VEGFR 2抑制剂ZM-323881仅在18%的SANC中抑制自发放电，表明VEGFR 1而不是VEGFR 2可能是基础心脏自律性的关键调节剂。 （三） 由于磷酸肌醇-3-激酶（PI 3-K）和磷脂酶C（PLC）都是EGFR和VEGFR的下游靶点，我们研究了这些靶点的抑制如何影响LCR参数和自发SANC放电。选择性PI 3-K抑制剂PI 103作用不明显，而PLC抑制剂U 73122能明显减少LCR的大小、数目，延长LCR周期，延长自发SANC周期。抑制基础PLC或PKC激活也明显抑制SANC的自发放电。 我们的结论是EGFR和VEGFR的基础活性参与了SANC在基础状态下正常自律性的调节。基础EGFR和VEGFR活性增加SANC的基础自发放电率;该效应通过Ca 2+时钟的支持来实现，即SR生成的LCR大小、每个自发周期的LCR数量增加以及预测自发SANC周期长度的LCR周期减少。

(1) We discovered that EGFR and VEGFR1/2 including their downstream targets PLC and PKC (assessed by RT-qPCR) are expressed in the SA node with the expression levels comparable to those in ventricle. (2) Specific EGFR inhibitor AG1478 markedly decreased spontaneous SANC beating rate (perforated patch-clamp technique) and this effect was largely reversible upon drug washout, indicating that EGFR is active in the basal state. Effects of AG1478 on spontaneous SANC firing were further clarified following investigation of the effects of EGFR inhibition on Ca2+ clock. AG1478 markedly suppressed SR Ca2+ cycling (confocal microscopy, Ca2+ indicator Fluo-3AM) in SANC, i.e. decreased the LCR size and number per each spontaneous cycle and prolonged the LCR period (the interval between AP-induced Ca2+ transient and subsequent LCR), which predicted the concomitant increase in the spontaneous cycle length. We also investigated effects of VEGFR1/2/3 inhibitor vatalanib on LCR characteristics in SANC. Vatalanib markedly suppressed LCR parameters, i.e., decreased the LCR size and number per each spontaneous cycle and prolonged the LCR period accompanied by increase in the spontaneous cycle length. Vatalanib also produced arrhythmic behavior of SANC. Overall, vatalanib reduced the spontaneous SANC beating rate by 50%. The selective VEGFR2 inhibitor ZM-323881, however, suppressed spontaneous firing in only 18% of SANC indicating that VEGFR1, but not VEGFR2, is likely the key modulator of basal cardiac automaticity. (3) Since both phosphoinositide-3-kinase (PI3-K) and phospholipase C (PLC) are downstream targets of EGFR and VEGFR we studied how inhibition of these targets would affect LCR parameters and spontaneous SANC firing. While selective PI3-K inhibitor PI-103 was lacking effects, PLC inhibitor U-73122, but not its inactive analog U-73343, markedly decreased the LCR size, number, extended the LCR period and prolonged the spontaneous SANC cycle length. Inhibition of basal PLC or PKC activation also markedly suppressed spontaneous firing of SANC. We conclude that basal activity of both EGFR and VEGFR are involved in regulation of normal automaticity of SANC in the basal state. Basal EGFR and VEGFR activity augment basal spontaneous firing rate of SANC; this effect is executed via support of Ca2+ clock, i.e. increase in SR-generated size of LCRs, number of LCR per each spontaneous cycle and decrease in the LCR period which predicts the spontaneous SANC cycle length.