Department of Physiology and HeartOtago, University of Otago, Dunedin, New Zealand (A.S.P., E.U.A., L.P.I.W., R.E.P., R.S.W., J.R.E.).
Department of Physiology, University of Auckland, New Zealand (A.S.P.).
Circ Res. 2023 Dec 8;133(12):1040-1055. doi: 10.1161/CIRCRESAHA.123.323571. Epub 2023 Nov 14.
Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca release via -nitrosylation of CaMKIIδ (Ca/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of -nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.
We measured Ca-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the -nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, -nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).
Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca transient response as well as increased Ca spark frequency. However, the increase in Ca spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.
We conclude that prior -nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that -nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.
一氧化氮（NO）已被确定为心脏β-肾上腺素能受体刺激过程中产生的信号分子。此外，NO 通过对 CaMKIIδ（钙/钙调蛋白激酶 II 德尔塔）的 - 亚硝基化触发自发 Ca 释放的作用也正在出现。NO 供体临床上常因其对心脏的心脏保护作用而使用，但尚不清楚 NO 供体如何调节促心律失常的 CaMKII 以改变心脏心律失常的发生率。我们在β-肾上腺素能受体刺激之前和期间，测试 CaMKIIδ 的 C 端 273 位半胱氨酸抑制位点和 C 端 290 位半胱氨酸激活位点的 - 亚硝基化在心脏 Ca 处理和心律失常发生中的作用。
我们测量了来自 C57BL/6J 野生型（WT）小鼠和缺乏 CaMKIIδ 表达（CaMKIIδ-KO）或 CaMKIIδ 的 - 亚硝基化位点在 C 端 273 位或 C 端 290 位缺失的小鼠（CaMKIIδ-C273S 和 -C290A 基因敲入小鼠）的分离心肌细胞中的 Ca 处理。将心肌细胞暴露于 NO 供体，包括 - 亚硝基谷胱甘肽（GSNO；150 μM）、硝普钠（200 μM）和β-肾上腺素能激动剂异丙肾上腺素（100 nmol/L）。
WT 和 CaMKIIδ-KO 心肌细胞均对异丙肾上腺素产生全效变力和变时 Ca 瞬变反应以及增加的 Ca 火花频率。然而，CaMKIIδ-KO 心肌细胞中 Ca 火花频率的增加明显减弱。GSNO 预处理可模拟 WT 心肌细胞中异丙肾上腺素诱导的 Ca 火花和波的保护作用，但在 CaMKIIδ-C273S 心肌细胞中失去这种作用。当异丙肾上腺素后应用 GSNO 时，WT 或 CaMKIIδ-C273S 中未观察到这种保护作用，但在 CaMKIIδ-C290A 中观察到。在 Langendorff 灌注分离的心脏中，GSNO 预处理可限制 WT 心脏中异丙肾上腺素诱导的心律失常，但不能限制 CaMKIIδ-C273S 心脏中的心律失常，而 GSNO 暴露后可维持或加重心律失常事件。
我们得出结论，CaMKIIδ 的 C 端 273 位的先前 - 亚硝基化可限制随后的β-肾上腺素能受体诱导的心律失常，但 C 端 290 位的 - 亚硝基化可能加重或维持β-肾上腺素能受体诱导的心律失常。这对 NO 供体在临床环境中的应用具有重要意义。
