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

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(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.