Department of Pharmacology, University of California, Davis, CA, USA.
Research Group in Veterinary Medicine, School of Veterinary Medicine, University Corporation Lasallista, Caldas, Antioquia, Colombia.
J Physiol. 2022 Nov;600(22):4865-4879. doi: 10.1113/JP283427. Epub 2022 Oct 30.
Cardiac mechanical afterload induces an intrinsic autoregulatory increase in myocyte Ca dynamics and contractility to enhance contraction (known as the Anrep effect or slow force response). Our prior work has implicated both nitric oxide (NO) produced by NO synthase 1 (NOS1) and calcium/calmodulin-dependent protein kinase II (CaMKII) activity as required mediators of this form of mechano-chemo-transduction. To test whether a single S-nitrosylation site on CaMKIIδ (Cys290) mediates enhanced sarcoplasmic reticulum Ca leak and afterload-induced increases in sarcoplasmic reticulum (SR) Ca uptake and release, we created a novel CRISPR-based CaMKIIδ knock-in (KI) mouse with a Cys to Ala mutation at C290. These CaMKIIδ-C290A-KI mice exhibited normal cardiac morphometry and function, as well as basal myocyte Ca transients (CaTs) and β-adrenergic responses. However, the NO donor S-nitrosoglutathione caused an acute increased Ca spark frequency in wild-type (WT) myocytes that was absent in the CaMKIIδ-C290A-KI myocytes. Using our cell-in-gel system to exert multiaxial three-dimensional mechanical afterload on myocytes during contraction, we found that WT myocytes exhibited an afterload-induced increase in Ca sparks and Ca transient amplitude and rate of decline. These afterload-induced effects were prevented in both cardiac-specific CaMKIIδ knockout and point mutant CaMKIIδ-C290A-KI myocytes. We conclude that CaMKIIδ activation by S-nitrosylation at the C290 site is essential in mediating the intrinsic afterload-induced enhancement of myocyte SR Ca uptake, release and Ca transient amplitude (the Anrep effect). The data also indicate that NOS1 activation is upstream of S-nitrosylation at C290 of CaMKII, and that this molecular mechano-chemo-transduction pathway is beneficial in allowing the heart to increase contractility to limit the reduction in stroke volume when aortic pressure (afterload) is elevated. KEY POINTS: A novel CRISPR-based CaMKIIδ knock-in mouse was created in which kinase activation by S-nitrosylation at Cys290 (C290A) is prevented. How afterload affects Ca signalling was measured in cardiac myocytes that were embedded in a hydrogel that imposes a three-dimensional afterload. This mechanical afterload induced an increase in Ca transient amplitude and decay in wild-type myocytes, but not in cardiac-specific CaMKIIδ knockout or C290A knock-in myocytes. The CaMKIIδ-C290 S-nitrosylation site is essential for the afterload-induced enhancement of Ca transient amplitude and Ca sparks.
心肌机械后负荷诱导肌球蛋白 Ca 动力学和收缩力的内在自身调节增加，以增强收缩（称为 Anrep 效应或缓慢力反应）。我们之前的工作表明，一氧化氮合酶 1（NOS1）产生的一氧化氮（NO）和钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）活性都是这种机械化学转导形式的必需介质。为了测试 CaMKIIδ（Cys290）上的单个 S-亚硝酰化位点是否介导增强的肌浆网 Ca 渗漏以及后负荷诱导的肌浆网（SR）Ca 摄取和释放增加，我们创建了一种新型基于 CRISPR 的 CaMKIIδ 基因敲入（KI）小鼠，该小鼠在 C290 处具有 Cys 到 Ala 的突变。这些 CaMKIIδ-C290A-KI 小鼠表现出正常的心脏形态和功能，以及基础肌球蛋白 Ca 瞬变（CaTs）和β-肾上腺素能反应。然而，NO 供体 S-亚硝基谷胱甘肽导致野生型（WT）肌球蛋白中 Ca 火花频率急性增加，而 CaMKIIδ-C290A-KI 肌球蛋白中则没有。使用我们的细胞凝胶系统在收缩过程中对肌球蛋白施加多轴三维机械后负荷，我们发现 WT 肌球蛋白表现出 Ca 火花和 Ca 瞬变幅度以及下降速率的后负荷诱导增加。这种后负荷诱导的作用在心脏特异性 CaMKIIδ 缺失和点突变 CaMKIIδ-C290A-KI 肌球蛋白中均被阻止。我们得出的结论是，C290 位点的 S-亚硝酰化对 CaMKIIδ 的激活对于介导肌浆网 Ca 摄取、释放和 Ca 瞬变幅度的固有后负荷诱导增强（Anrep 效应）是必不可少的。该数据还表明，NOS1 激活位于 CaMKII 上 C290 的 S-亚硝酰化的上游，并且这种分子机械化学转导途径有利于使心脏增加收缩力，以限制主动脉压（后负荷）升高时心搏量的减少。关键点：创建了一种新型基于 CRISPR 的 CaMKIIδ 基因敲入小鼠，其中阻止了 Cys290（C290A）处 S-亚硝酰化对激酶的激活。在嵌入迫使产生三维后负荷的水凝胶中的心肌细胞中测量了后负荷如何影响 Ca 信号。这种机械后负荷诱导野生型肌球蛋白的 Ca 瞬变幅度和衰减增加，但在心脏特异性 CaMKIIδ 缺失或 C290A 基因敲入肌球蛋白中则没有。CaMKIIδ-C290 S-亚硝酰化位点对于后负荷诱导的 Ca 瞬变幅度和 Ca 火花增强是必需的。
