A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2+ transients and contractile function in cardiomyocytes.
A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2+ transients and contractile function in cardiomyocytes.
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DOI：
10.1016/j.yjmcc.2014.09.006
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发表时间：
2014-11
影响因子：
5
通讯作者：
Hiroki Sakai;Y. Ikeda;Takeshi Honda;Yoshie Tanaka;K. Shiraishi;M. Inui
中科院分区：
文献类型：
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作者：
Hiroki Sakai;Y. Ikeda;Takeshi Honda;Yoshie Tanaka;K. Shiraishi;M. Inui
肌浆网的肌浆网Ca ~（2+）-ATP酶2a（SERCA 2a）-受磷蛋白（PLN）系统在心室肌细胞内Ca ~（2+）循环的调节中起关键作用。鉴于Ca 2+循环在心力衰竭中受损，有助于这一过程的蛋白质是治疗这种疾病的潜在靶点。我们现在已经分离出PLN特异性的适配体与硫代磷酸酯修饰的骨干从RNA分子库包含一个随机的40个核苷酸的序列，通过应用配体的系统进化指数富集（SELEX）协议与融合蛋白，含有人PLN的胞质区域。这些适体之一被缩短为30个核苷酸的寡聚体（RNA-Apt 30）而不丧失功能。RNA-Apt 30对PLN的胞质区域显示出高亲和力（Kd= 11 nM），但它不结合PLN的磷酸化形式或磷酸化模拟物突变体。它还通过减轻PLN介导的抑制作用增加了分离的心脏SR囊泡中的SERCA 2a活性，EC 50为18 nM。结合RNA-Apt 30的细胞穿透肽允许其交付到成年大鼠心肌细胞，其中它增强了Ca 2+瞬变和收缩功能。在β-肾上腺素能受体拮抗剂普萘洛尔存在下，适体的这些作用也很明显。因此，这种细胞穿透PLN适体可以为开发新的心力衰竭治疗药物提供基础，而不需要基因转移或内源性蛋白质表达的变化。
The sarco(endo)plasmic reticulum Ca2 +-ATPase 2a (SERCA2a)–phospholamban (PLN) system of sarcoplasmic reticulum plays a pivotal role in regulation of intracellular Ca2 +cycling in ventricular cardiomyocytes. Given that Ca2 +cycling is impaired in heart failure, proteins that contribute to this process are potential targets for the treatment of this condition. We have now isolated PLN-specific aptamers with a phosphorothioate-modified backbone from a library of RNA molecules containing a randomized 40-nucleotide sequence by application of the systematic evolution of ligands by exponential enrichment (SELEX) protocol with a fusion protein containing the cytoplasmic region of human PLN. One of these aptamers was shortened to a 30-nucleotide oligomer (RNA-Apt30) without loss of function. RNA-Apt30 showed a high affinity for the cytoplasmic region of PLN (Kd= 11 nM), but it did not bind to the phosphorylated form of PLN or to a phosphomimetic mutant. It also increased SERCA2a activity in isolated cardiac SR vesicles with an EC50of 18 nM by relieving PLN-mediated inhibition. Conjugation of RNA-Apt30 to a cell-penetrating peptide allowed its delivery into adult rat cardiomyocytes, in which it enhanced both Ca2 +transients and contractile function. These effects of the aptamer were also apparent in the presence of the β-adrenergic receptor antagonist propranolol. This cell-penetrating PLN aptamer may thus provide a basis for the development of new therapeutic agents for heart failure without the need for gene transfer or a change in endogenous protein expression.