Protein Kinase Novel 2 (PKN2) in heart

项目摘要 新型蛋白激酶（PKN）激酶家族，也称为蛋白激酶C相关激酶，属于 PKC超家族Pkn 2基因座的单核苷酸多态性与以下风险相关： 冠状动脉疾病/心肌梗死和PKN 2蛋白水平升高与心脏 疾病，突出PKN 2在心脏中的重要性。小鼠中Pkn 2的整体缺失导致致死性， 胚胎第10天，有心脏缺陷。利用SM 22 α-Cre小鼠，Pkn 2的条件性缺失导致 E13.5和断奶之间的部分致死性，存活的突变体显示异常的心脏表型。 然而，这些观察结果强烈表明PKN 2在发育中的心脏中起着关键作用，因为 SM 22 α-Cre不仅在早期发育的心肌细胞中表达，而且在平滑肌细胞、骨骼肌细胞、平滑肌细胞 肌肉，骨髓和淋巴免疫细胞，这项先前的研究没有解决心肌细胞- 具体要求。有趣的是，最近的一项研究表明，成年人Pkn 1和Pkn 2的缺失 心肌细胞，利用αMHC-MerCreMer小鼠，不影响基础心脏功能，但保护小鼠免受 压力超负荷和血管紧张素II诱导的心脏肥大和心力衰竭，表明PKN 失活可能是心力衰竭独特的治疗靶点。局部的矛盾 SM 22 α-Cre：Pkn 2基因敲除小鼠的胚胎致死率和成年α-MHC- MerCreMer：Pkn 1/2双敲除小鼠强调了定义PKN 2在以下方面的潜在作用的迫切需要： 不同发育阶段的心肌细胞。为了解决这一矛盾，我们创造了新的 Pkn 2心肌细胞特异性组成型敲除（cKO）和Pkn 2他莫昔芬诱导的心肌细胞特异性 分别利用Xmlc 2-Cre和Tnnt 2-MerCreMer小鼠系的敲除（icKO）小鼠模型。后 初步表征，Pkn 2 cKO小鼠显示出部分出生后致死性和心脏形态学变化。 早在E12.5就有缺陷。对存活突变体的超声心动图研究显示是扩张型心肌病 在1月龄和3月龄时Pkn 2 cKO突变体中的表型。与公布的报告相反， pkn 2在成年心肌细胞中不影响基础心功能，我们的初步观察表明， pkn 2缺乏对发育中的心肌细胞是有害的。综上所述，上述证据使我们 PKN 2在心肌细胞发育的不同阶段通过心肌细胞的生长发育发挥不同作用的假说， 特异性底物的磷酸调节。因此，我们的具体目标是：1。为了阐明PKN 2在 通过分析Pkn 2 cKO小鼠的心脏和心肌细胞结构和功能， 利用无偏磷酸化蛋白质组学鉴定心肌细胞中PKN 2的内源性底物， 用类似物敏感的PKN 2突变体的化学遗传学方法，以及2.为了确定心肌细胞- 通过分析Pkn 2 icKO小鼠在出生后发育中对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.