A novel gene and mechanisms for statin-induced myopathy in the mouse

摘要/摘要 降胆固醇的他汀类药物显著降低心血管发病率和死亡率，但 他汀类药物使用者会经历肌肉疼痛/虚弱，这限制了他汀类药物在这些人中的应用。更好的 了解他汀类药物诱导的肌病的遗传易感性将促进我们对 并提出了预防和治疗这些症状的新策略。此应用程序构建在 我们发现了一个影响他汀类药物诱导的肌病易感性的新的遗传基因。使用 94个不同的近交系小鼠品系的独特遗传参照组，我们评估了 辛伐他汀在800多只单独的小鼠中使用。我们对全基因组关联的综合分析(GWAS)和 表达定量基因座(EQTL)数据证实TPMT是一种高概率随机基因，其特异性 与他汀类药物引起的肌病有关。TPMT基因编码硫代嘌呤S甲基转移酶 (TPMT)，代谢硫代嘌呤类药物等外源物质；唯一已知的内源性底物是 硒半胱氨酸是一种氨基酸，选择性地结合到硒蛋白中，硒蛋白在 维持细胞氧化还原状态。硒蛋白的合成需要产生异戊烯基中间体 通过HMG辅酶A途径合成胆固醇，并在他汀类药物存在下被还原。 有趣的是，某些硒蛋白不足会引起类似他汀类药物引起的肌肉症状。 肌病。我们假设TPMT表达水平的遗传变异与他汀类药物相结合 治疗构成了他汀类肌病诱导的两次打击机制：(1)他汀类药物限制供应 和(2)导致TPMT水平升高的基因变异 减少蛋白质合成所需的硒半胱氨酸库。我们将使用一个 体内和体外研究相结合。在目标1中，我们将确定调制TPMT水平的效果 他汀类药物通过腺相关病毒(AAV)介导的功能获得和丧失途径治疗小鼠 基因转移到我们定义为肌病易感或抗病的遗传背景中。转换 肌病敏感性和抵抗之间的关系将提供强有力的证据表明TPMT是一种他汀类肌病 易感基因。在目标2中，我们将研究他汀类药物-TPMT-硒蛋白轴的每个成分 他汀类肌病的潜在贡献。我们将首先评估TPMT表达水平的调节 改变体内和培养的肝细胞中的硒蛋白水平。接下来我们将确定硒蛋白 他汀类药物水平影响他汀类药物对培养肌管的肌毒性作用。最后，我们将评估 他汀类/硒蛋白对心肌细胞线粒体功能和活性氧水平的影响。完成 我们的目标将有助于深入了解他汀类肌病的遗传易感性和发病机制，并建议 个体化他汀类药物治疗的策略。

SUMMARY/ABSTRACT Cholesterol-lowering statin drugs significantly reduce cardiovascular morbidity and mortality, but a subset of statin users experience muscle pain/weakness, which limits the utility of statins in these individuals. A better understanding of the genetic predisposition to statin-induced myopathy will advance our understanding of the pathogenesis and also suggest new strategies to prevent and treat these symptoms. This application builds on our identification of a novel genetic locus that influences susceptibility to statin-induced myopathy. Using a unique genetic reference panel of 94 diverse inbred mouse strains, we assessed the adverse effects of simvastatin in more than 800 individual mice. Our integrated analysis of genome-wide association (GWAS) and expression quantitative loci (eQTL) data identified Tpmt as a high-probability casual gene that is specifically associated with statin-induced myopathy. The Tpmt gene encodes the enzyme thiopurine S-methyltransferase (TPMT), which metabolizes xenobiotics such as thiopurine drugs; the only known endogenous substrate is selenocysteine, an amino acid that is selectively incorporated into selenoproteins, which have critical roles in maintaining cellular redox state. Selenoprotein synthesis requires isopentenyl intermediates that are produced by the HMG CoA pathway for cholesterol synthesis, and which are reduced in the presence of statins. Interestingly, insufficiency of some selenoproteins causes muscle symptoms that resemble statin-induced myopathy. We hypothesize that genetic variation in Tpmt expression levels in combination with statin treatment constitutes a two-hit mechanism for the induction of statin myopathy: (1) statin limits the supply of isopentenyl groups for selenocysteine synthesis, and (2) genetic variation causing elevated TPMT levels reduces the pool of selenocysteine available for protein synthesis. We will test our hypothesis using a combination of in vivo and in vitro studies. In Aim 1, we will determine the effect of modulating Tpmt levels in statin-treated mice through gain- and loss-of-function approaches using adeno-associated virus (AAV)-mediated gene transfer into genetic backgrounds that we have defined as myopathy-susceptible or -resistant. Conversion between myopathy sensitivity and resistance will provide strong evidence that Tpmt is a statin myopathy susceptibility gene. In Aim 2, we will investigate each component of the statin–TPMT–selenoprotein axis for potential contributions to statin myopathy. We will first assess whether modulation of Tpmt expression levels alters selenoprotein levels in vivo and in cultured hepatocytes. We will next determine whether selenoprotein levels influence statin-induced myotoxicity in cultured myotubes. Finally, we will assess the effects of statin/selenoproteins on myocyte mitochondrial function and reactive oxygen species levels. The completion of our aims will contribute insights into the genetic susceptibility and pathogenesis of statin myopathy, and suggest strategies for personalized statin therapy.