Protein Kinase Novel 2 (PKN2) in heart

项目总结 蛋白激酶新奇(PKNS)家族，又称蛋白激酶C相关蛋白，属于 PKC超级家族。PKN2基因座的单核苷酸多态与更大的风险相关 冠状动脉疾病/心肌梗死和PKN2蛋白水平升高与心脏相关 疾病，强调了PKN2在心脏中的重要性。小鼠体内PKN2基因的全局缺失导致致死率 胚胎第10天(E)合并心脏缺陷。利用Sm22α-Cre小鼠，有条件地删除PKN 2，导致 E13.5和断奶之间的部分致死性，存活的突变体表现出异常的心脏表型。 然而，这些观察结果有力地表明，PKN2在心脏发育中起着关键作用，因为 Sm22α-Cre不仅在发育早期的心肌细胞中表达，而且在平滑肌、骨骼肌中也有表达 肌肉，髓系和淋巴免疫细胞，这项先前的研究没有涉及心肌细胞- 具体要求。有趣的是，最近的一项研究表明，在成年人中，PKN1和PKN2的缺失 利用αMHC-Mercremer小鼠的心肌细胞不影响基本的心功能，但保护小鼠免受 压力超负荷和血管紧张素II诱导的心肌肥厚和心力衰竭，提示PKN 失活可能是心力衰竭的一个独特的治疗靶点。局部与局部之间的矛盾 Sm22α-Cre：PKN_2基因敲除小鼠的胚胎致死性及成年α小鼠的保护作用 MerCreMer：PKN1/2双基因敲除小鼠强调了定义PKN2在 不同发育阶段的心肌细胞。为了解决这一矛盾，我们产生了小说 心肌细胞特异性基因敲除(CKO)和三苯氧胺诱导的心肌细胞特异性基因敲除 分别利用Xmlc2-Cre和Tnnt2-MerCreMer小鼠品系建立基因敲除(IcKO)小鼠模型。vt.在.的基础上 初步鉴定，PKN2 CKO小鼠表现出部分出生后致死性和心脏形态 早在E12.5就存在缺陷。存活突变的超声心动图研究显示扩张型心肌病 1个月龄和3个月龄突变体的表型。与已公布的报告相比，损失 成人心肌细胞中的PKN2不影响基础心功能，我们的初步观察表明 在发育中的心肌细胞中，PKN2的缺乏是有害的。综上所述，上述证据使我们能够 PKN2在心肌细胞发育的不同阶段发挥不同作用的假说 特定底物的磷调节。因此，我们的具体目标是：1.阐明PKN_2在 通过对PKN2 CKO小鼠心肌和心肌细胞结构和功能的分析，对心肌细胞进行了研究 利用无偏向磷酸化蛋白质组学和核糖核酸技术鉴定心肌细胞内源性蛋白激酶2的底物 化学遗传学方法与类似敏感的PKN2突变体，以及2.为了确定心肌细胞- 通过对PKN_2 icKO小鼠的分析，发现其在出生后发育中对PKN_2的特异性需求。

PROJECT SUMMARY The Protein Kinase Novel (PKNs) family of kinases, also known as Protein Kinase C-related kinases, belong to the PKC superfamily. A single nucleotide polymorphism at the Pkn2 locus is associated with greater risk for coronary artery disease/myocardial infarction, and elevated levels of PKN2 protein are associated with heart disease, highlighting the importance of PKN2 in heart. Global deletion of Pkn2 in mouse results in lethality at embryonic day (E) 10 with cardiac defects. Conditional deletion of Pkn2, utilizing SM22α-Cre mice, results in partial lethality between E13.5 and weaning, with surviving mutants displaying abnormal cardiac phenotypes. These observations strongly suggest that PKN2 plays a critical role in the developing heart, however, because SM22α-Cre is expressed not only in early developing cardiomyocytes, but also in smooth muscle, skeletal muscle, and myeloid and lymphoid immune cells, this previous study does not address the cardiomyocyte- specific requirement. Intriguingly, a recent study showed that deletion of Pkn1 and Pkn2 in adult cardiomyocytes, utilizing αMHC-MerCreMer mice, did not affect basal cardiac function, but protected mice from pressure overload- and angiotensin II-induced cardiac hypertrophy and heart failure, suggesting that PKN inactivation could be a unique therapeutic target for heart failure. The contradiction between the partial embryonic lethality of SM22α-Cre:Pkn2 knockout mice and protective effects observed in adult αMHC- MerCreMer:Pkn1/2 double knockout mice highlights a critical need to define potential roles of PKN2 in cardiomyocytes at different developmental stages. To address this contradiction, we have generated novel Pkn2 cardiomyocyte-specific constitutive knockout (cKO) and Pkn2 tamoxifen-inducible cardiomyocyte-specific knockout (icKO) mouse models utilizing Xmlc2-Cre and Tnnt2-MerCreMer mouse lines, respectively. Upon preliminary characterization, Pkn2 cKO mice displayed partial postnatal lethality and cardiac morphological defects as early as E12.5. Echocardiographic studies of surviving mutants revealed a dilated cardiomyopathy phenotype in Pkn2 cKO mutants at both 1 and 3 months of age. In contrast to published reports that loss of Pkn2 in adult cardiomyocytes does not affect basal cardiac function, our preliminary observations suggest that Pkn2 deficiency in developing cardiomyocytes is detrimental. Taken together, the above evidence leads us to the hypothesis that PKN2 plays distinct roles at different stages of cardiomyocyte development through the phosphoregulation of specific substrates. Accordingly, our specific aims are: 1. To elucidate the role of PKN2 in cardiomyocytes by analysis of cardiac and cardiomyocyte structure and function in Pkn2 cKO mice, and to identify endogenous substrates of PKN2 in cardiomyocytes by utilizing unbiased phosphoproteomics and a chemical-genetics approach with an analog-sensitive PKN2 mutant, and 2. To determine the cardiomyocyte- specific requirement for PKN2 in postnatal development by analysis of Pkn2 icKO mice.