1 Division of Cardiology, Johns Hopkins Medical Institutions , Baltimore, Maryland.
Antioxid Redox Signal. 2013 Oct 10;19(11):1185-97. doi: 10.1089/ars.2012.5057.
Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts.
Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available.
HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a.
PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.
亚硝酰（HNO）与硫醇相互作用，作为蛋白质功能的氧化还原敏感调节剂。它增强肌浆网 Ca（2+）摄取和肌丝 Ca（2+）敏感性，改善心脏收缩力。这种活性导致了 HNO 供体在心力衰竭中的临床测试。在这里，我们测试了 HNO 是否以氧化还原依赖的方式改变磷蛋白（PLN）和肌浆网 Ca（2+）-ATP 酶（SERCA2a）之间的抑制相互作用，从而改善分离的心肌细胞/心脏中的 Ca（2+）处理。
从对照（野生型[WT]）或 PLN 敲除（pln（-/-））小鼠中分离心室细胞、肌浆网（SR）囊泡和整个心脏。与 WT 相比，pln（-/-）心肌细胞显示出增强的静息肌节缩短、峰值 Ca（2+）瞬变和减弱的β肾上腺素能反应性。HNO 刺激 WT 心肌细胞缩短、松弛和 Ca（2+）瞬变，并在完整心脏中引起正性变力/变时性。这些变化在 pln（-/-）细胞/心脏中明显减弱。HNO 增强了 WT 的 SR Ca（2+）摄取，但不是 pln（-/-）的 SR 囊泡。在表达 SERCA2a±PLN 的昆虫细胞微粒体中的光谱研究表明，HNO 增加了 Ca（2+）依赖性 SERCA2a 的构象灵活性，但只有当 PLN 存在时才会增加。在心肌细胞中，HNO 通过稳定 PLN 处于二硫键依赖性的寡聚构象来实现这种作用，减少了可用的游离抑制性单体 PLN 的量。
肌细胞 PLN 寡聚化的 HNO 依赖性氧化还原变化减轻了 PLN 对 SERCA2a 的抑制。
PLN 在 HNO 诱导的 SERCA2a 活性增强中起核心作用，导致完整心肌细胞和心脏的变力/变时性增加。PLN 仍然与 SERCA2a 物理相关；然而，可用的单体 PLN 较少，导致酶的抑制作用降低。这些发现为改善衰竭心脏中的 Ca（2+）处理提供了新的途径。
