title: A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2+ transients and contractile function in cardiomyocytes.
abstract: The sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-phospholamban (PLN) system of sarcoplasmic reticulum plays a pivotal role in regulation of intracellular Ca(2+) cycling in ventricular cardiomyocytes. Given that Ca(2+) 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 EC50 of 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 Ca(2+) 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.
authors.affiliation: Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakai
authors.affiliation: Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.affiliation: Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Shiraishi
authors.affiliation: Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan. Electronic address: minui@yamaguchi-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Inui
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D020013:Calcium Signaling / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D004285:Dogs; D008297:Male; D008969:Molecular Sequence Data; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D011817:Rabbits; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D052157:Aptamers, Nucleotide; D002135:Calcium-Binding Proteins; D057846:Cell-Penetrating Peptides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Aptamer; Calcium signaling; Cardiac sarcoplasmic reticulum; Cardiomyocyte; Cell-penetrating peptide; Phospholamban