Cardiac Oxidative Signaling and Physiological Hypertrophy in the Na/K-ATPase a1s/sa2s/s Mouse Model of High Affinity for Cardiotonic Steroids

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase a1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase a1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, a1, a2 and ß1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3–6 months, increased a1 sensitivity to CTS resulted in a significant increase of cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio, accompanied by an increase of myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the a1r/ra2s/s mouse failed to do so in the a1s/sa2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase a1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions. Overall design: Whole heart mRNA from 3-month old Na/K-ATPase a1r/ra2s/s (resistant) and Na/K-ATPase a1s/sa2s/s (sensitive) mice.

钠钾ATP酶（Na/K-ATPase）是哇巴因、地高辛等强心甾类化合物（cardiotonic steroids, CTS）的特异性受体。在用于心脏疾病治疗的药理学浓度下，CTS可抑制钠钾ATP酶的离子泵功能。而在远低于血液中内源性CTS报道浓度的水平下，它们可通过启动钠钾ATP酶介导、活性氧（reactive oxygen species, ROS）依赖的信号通路，刺激培养的心肌细胞发生肥厚性生长。
为探究内源性浓度的CTS在体内对心脏结构与功能的潜在影响，我们对比了两类小鼠：一类表达天然抗性型钠钾ATP酶α1亚基，另一类为同龄基因工程改造小鼠，其表达对CTS具有高亲和力的突变型钠钾ATP酶α1亚基。
该模型中，心脏总钠钾ATP酶活性、α1、α2及β1蛋白含量均未发生改变，且心脏钠钾ATP酶对哇巴因的剂量反应曲线如预期般左移。在3~6月龄的雄性小鼠中，α1亚基对CTS的敏感性升高会导致心脏羰基化蛋白含量显著升高，提示活性氧生成增加。同时可检测到心脏重量/胫骨长度比值出现约15%的中度但显著升高，并伴随心肌细胞横截面积增大。超声心动图分析未发现心脏功能出现任何改变，且未出现纤维化或胎儿基因程序的重新激活。
RNA测序（RNA sequencing）分析显示，与能量代谢相关的通路出现上调，而与细胞外基质组织相关的通路则出现下调。与后者的功能作用一致，在α1^r/rα2^s/s小鼠中可触发纤维化的血管紧张素II（angiotensin-II）刺激，在α1^s/sα2^s/s小鼠中无法诱导纤维化。
综上，上述结果表明，在实验室基础饲养条件下的小鼠体内，循环CTS、钠钾ATP酶α1亚基、活性氧与生理性心肌肥厚之间存在关联。整体实验设计：取自3月龄钠钾ATP酶α1^r/rα2^s/s（抗性型）与α1^s/sα2^s/s（敏感型）小鼠的全心脏mRNA样本。