Division of Cardiovascular Medicine, Radcliffe Department of Medicine (J.N.S., B.V., S.M.C., N.R., O.L., G.A.M., P.R.G., R.J., K.M.C., B.C.), University of Oxford, United Kingdom.
Department of Physiology, Anatomy and Genetics (S.M., P.S., M.Z.), University of Oxford, United Kingdom.
Circulation. 2021 Feb 2;143(5):449-465. doi: 10.1161/CIRCULATIONAHA.120.046761. Epub 2020 Nov 13.
Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored.
Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo.
In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, =0.023; 2.4-fold in mice, <0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, <0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, <0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion.
Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.
激酶氧化是一种关键的信号机制，通过改变细胞内的氧化还原状态来改变心脏功能。在心肌中，PKARIα（I 型蛋白激酶 A）可以被可逆氧化，在全酶复合物中形成蛋白间二硫键。然而，PKARIα 二硫键形成对心脏下游信号的影响，特别是在缺血再灌注（I/R）等氧化应激状态下，仍未得到探索。
使用心肺旁路和再灌注前后患者的心房组织以及 I/R 或假手术处理的小鼠的左心室（LV）组织，通过免疫印迹评估 PKARIα 二硫键的形成。为了确定二硫键形成对 PKARIα 催化活性和亚细胞定位的影响，在来自“氧化还原死亡”（Cys17Ser）PKARIα 基因敲入小鼠及其野生型同窝仔鼠的成纤维细胞、乳鼠心肌细胞或成年 LV 心肌细胞中进行活细胞荧光成像和受激发射损耗超分辨率显微镜检查。通过在 fura2 负载的 LV 心肌细胞中评估基因型之间的细胞内钙动力学，以及在体外评估 I/R 损伤，比较基因型之间的差异。
在人和小鼠中，发现心肌 PKARIα 二硫键形成显著增加（在人类中增加 2 倍，=0.023；在小鼠中增加 2.4 倍，<0.001），对体内 I/R 有反应。在小鼠 LV 心肌细胞中，发现含有二硫键的 PKARIα 不会影响催化活性，而是导致 AKAP（A-激酶锚定蛋白）结合增强，全酶优先定位于溶酶体。PKARIα 通过氧化还原依赖性调节溶酶体双孔通道足以防止 LV 心肌细胞从肌浆网中释放全局钙，而不影响内在的ryanodine 受体渗漏或磷酸化。在“氧化还原死亡”基因敲入小鼠心脏中，I/R 诱导的 PKARIα 二硫键形成缺失导致梗死面积增加（增加 2 倍，<0.001），并伴随 LV 收缩功能恢复减少（减少 1.6 倍，<0.001），在再灌注时给予溶酶体双孔通道抑制剂 Ned-19 可预防这种情况。
二硫键修饰将 PKARIα 靶向溶酶体，在那里它充当双孔通道介导的全局钙释放触发的守门员。在缺血后心脏中，这种调节机制对于防止广泛损伤至关重要，并为设计心脏保护治疗提供了一个新的靶点。
