The sinoatrial node (SAN) is the origin of the electrical signals for rhythmic heartbeats in mammals. The spontaneous firing of SAN pacemaker cells (SANPCs) triggers cardiac contraction. ‘Local Ca²⁺ release’ (LCR), a unique cellular activity, acts as the ‘engine’ of the spontaneous firing of SANPCs. However, the mechanism of LCR initiation remains unclear. Here, we report that endogenous glutamate drives LCRs in SANPCs. Using a glutamate sensor, we unraveled a tight correlation between glutamate accumulation and LCR occurrence, indicating a potential relationship between glutamate and LCRs. Intracellular application of glutamate significantly enhanced the LCRs in both intact and permeabilized SANPCs. Mechanistically, we revealed that mitochondrial excitatory amino acid transporter 1 (EAAT1)-dependent mitochondrial glutamate import promoted ROS generation, which in turn led to the oxidation of Ca²⁺-handling proteins, ultimately resulting in enhanced LCRs. Importantly, EAAT1 depletion reduced both the spontaneous firing rates of isolated SANPCs and the heart rate in vitro and in vivo, suggesting the central role of EAAT1 as a glutamate transporter in the regulation of cardiac autonomic rhythm. In conclusion, our results indicate that glutamate serves as an LCR igniter in SANPCs, adding a potentially important element to the coupled-clock theory that explains the origin of spontaneous firing. These findings shed new light on the future prevention and treatment of cardiac pacemaker cell-related arrhythmias.

窦房结 (SAN) 是哺乳动物节律性心跳电信号的来源。SAN 起搏细胞 (SANPC) 的自发放电触发心脏收缩。'本地 Ca ²⁺释放”(LCR) 是一种独特的细胞活动，充当 SANPC 自发放电的“引擎”。然而，LCR 启动的机制仍不清楚。在这里，我们报告内源性谷氨酸驱动 SANPC 中的 LCR。使用谷氨酸传感器，我们揭示了谷氨酸积累与 LCR 发生之间的紧密相关性，表明谷氨酸和 LCR 之间存在潜在关系。谷氨酸的细胞内应用显着增强了完整和透化 SANPC 中的 LCR。从机制上讲，我们发现线粒体兴奋性氨基酸转运体 1 (EAAT1) 依赖性线粒体谷氨酸导入促进了 ROS 的生成，进而导致 Ca ^2+的氧化-处理蛋白质，最终导​​致 LCR 增强。重要的是，EAAT1 耗尽降低了分离的 SANPC 的自发放电率以及体外和体内的心率，表明 EAAT1 作为谷氨酸转运蛋白在调节心脏自主节律中的核心作用。总之，我们的结果表明谷氨酸在 SANPC 中充当 LCR 点火器，为解释自发放电起源的耦合时钟理论添加了一个潜在的重要元素。这些发现为未来预防和治疗心脏起搏器细胞相关的心律失常提供了新的思路。