Effect of cardiomyocyte specific KO of AKT1 + AKT2 on gene expression profile of the heart

In the mammalian heart AKT1 and AKT2 are the isoforms of the protein kinase AKT (protein kinase B) which are predominantly expressed. AKT isoforms exert common and specific functions in the field of metabolism, cellular growth, apoptosis and cell migration. To identify specific and common functions of AKT1 and AKT2 isoforms, we generated tamoxifen-inducible, cardiomyocyte-specific AKT1+AKT2 double knockout mice (iCMAKT – KO mice). Inactivation of AKT isoforms was achieved by application of 4-OH-tamoxifen, which activates an OH-Tx inducible cre-recombinase expressed under the control of the αMHC-promoter (αMHC-mercremer). Transgenic mice expressing only the αMHCmercremer construct were also treated with OH-Tx and served as controls. To identify alterations in cardiac gene expression due to AKT deletion we analyzed gene expression profiles of control hearts and iCMAKT1+2 hearts. Gene expression of mouse hearts of inducible, cardiomyocyte-specific AKT1+AKT2 KO mice. Mice express a mercremer recombinase under control of the αMHC promotor. AKT1 and AKT2 genes have loxP-flanked exons6 and7 and exon 5 exon 6 , respectively. Control mice only express the MHCmercremer gene. For RNA isolation four hearts of control control mice and three double KO were used. At age of 3 month mice were treated with 500µg hydroxytamoxifen (OH-Tx) per day for 5 days to induce the specific KO of AKT alleles in cardiomyocytes. Control mice express the MHCmercremer construct and were treated with OH-Tx equally. For RNA isolation four hearts of control and KO mice were harvested from 3 month old male mice 10 days after induction with OH-Tx. RNA was isolated from heart apex. 3 month old male mice after 5 injections of 4OH-tamoxifen served as controls.

在哺乳动物心脏中，AKT（蛋白激酶B，protein kinase B）的主要表达同工型为AKT1与AKT2。AKT同工型在代谢调控、细胞生长、细胞凋亡及细胞迁移过程中兼具共通功能与特异性功能。为鉴定AKT1与AKT2同工型的特异性及共通功能，我们构建了他莫昔芬（tamoxifen）诱导型、心肌细胞特异性AKT1+AKT2双基因敲除小鼠（iCMAKT-KO小鼠）。通过施加4-羟基他莫昔芬（4-OH-tamoxifen）可实现AKT同工型的基因失活：该化合物可激活受α肌球蛋白重链启动子（αMHC-promoter）调控表达的OH-Tx诱导型Cre重组酶（cre-recombinase）。仅表达αMHCmercremer构建体的转基因小鼠同样经OH-Tx处理，作为阴性对照。为探究AKT敲除引发的心脏基因表达变化，我们分析了对照心脏与iCMAKT1+2心脏的基因表达谱。本数据集对应诱导型心肌细胞特异性AKT1+AKT2双敲除小鼠的心脏基因表达情况：小鼠体内表达受αMHC启动子调控的mercremer重组酶；AKT1与AKT2基因分别带有loxP侧翼的外显子6、7与外显子5、6；对照小鼠仅表达MHCmercremer基因。实验中用于RNA分离的样本包括4只对照小鼠心脏与3只双敲除小鼠心脏。3月龄小鼠每日经500μg羟基他莫昔芬（OH-Tx）处理，连续给药5天，以诱导心肌细胞内AKT等位基因的特异性敲除。对照小鼠同样表达MHCmercremer构建体，并接受同等剂量的OH-Tx处理。于OH-Tx诱导10天后，从3月龄雄性小鼠体内采集对照与敲除小鼠的心脏样本，样本取自心尖部位用于RNA分离。经5次4-羟基他莫昔芬注射的3月龄雄性小鼠作为对照分组。