miR-1 Overexpression Enhances Ca 2 (cid:1) Release and Promotes Cardiac Arrhythmogenesis by Targeting PP2A Regulatory Subunit B56 (cid:2) and Causing CaMKII-Dependent Hyperphosphorylation of RyR2

—MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation–contraction coupling and Ca 2 (cid:1) cycling in rat ventricular myocytes using methods of electrophysiology, Ca 2 (cid:1) imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca 2 (cid:1) current, flattening of Ca 2 (cid:1) transients voltage dependence, and enhanced frequency of spontaneous Ca 2 (cid:1) sparks while reducing the sarcoplasmic reticulum Ca 2 (cid:1) content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1 –overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca 2 (cid:1) , events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca 2 (cid:1) /calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56 (cid:1) involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation–contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels. ( Circ Res . 2009;104:514-521.) vector following the manufacturer’s protocol (Invitrogen) after treating the PCR 10 minutes with Taq polymerase. Plasmid DNA was subsequently isolated from recombinant colonies and sequenced to ensure authenticity. The B56 α 3 ′ -UTR inserts were removed from the pCR™2.1 plasmid by Eco RI digestion. The fragments were subsequently gel purified, filled in and blunt-end ligated into a filled-in XhoI site which is located downstream of the Renilla luciferase (r-luc) reporter gene (psiCHECK-2™, Promega). The promoter used for Renilla luciferase expression in the siCHECK vector is the SV40 promoter. Additionally, the psiCHECK-2 vector possesses a secondary firefly reporter expression cassette which is under the control of the HSV-TK promoter. This firefly reporter cassette has been specifically designed to be an intraplasmid transfection normalization reporter; thus when using the psiCHECK-2 vector, the Renilla luciferase signal is normalized to the firefly luciferase signal. The authenticity and orientation of the inserts relative to the Renilla luciferase gene were confirmed by sequencing. The resulting recombinant plasmid was designated, psiCHEK-B56 α . Transformed bacterial cultures were grown and each reporter construct (psiCHECK or psiCHECK-B56 α ) was purified by using PureLink™ Hipure Plasmid Maxiprep Kit (Invitrogen). the cells after treatment and the samples were instantly frozen in liquid N2. Cell lysate proteins were subjected to 4% to 20% SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad Labs, CA). Primary antibodies used were: anti-PLB and anti-SERCA2a (both a gift from Dr. M. Periasamy, The Ohio State Univ., OH); anti-phospho-PLB- T17, anti-phospho-PLB-S16 (both from Badrilla, UK); anti-phospho-RyR2-S2808, anti-phospho-RyR2-S2814 (both a gift from Dr. A.R. Marks, Columbia Univ., NY); anti-phospho-RyR2-S2030(a gift from Dr. H.H. Valdivia, Univ. of Wisconsin, WI); anti-RyR2, anti-CASQ2, anti-CAMKII Tr-287 and anti-NCX1 (all from ABR, CO); anti-PP2A B56 α , anti-PP1 (both from Upstate, NY); anti-PP2AC (from Calbiochem); anti-CaMKII (from Santa Cruz Biotech., CA); anti-Ca v 1.2 (from Alomone, Israel). Protein bands were visualized using the Super Signal West Pico kit (PIERCE, IL). Phosphorylation analysis for Ca v 1.2 ( α 1C) was carried out using Pro-Q Diamond stain technology. In brief, protein gel was fixed overnight and stained using the Pro-Q Diamond phosphoprotein basic staining protocol (Invitrogen Corp., CA). The gel was imaged in a Bio-Rad Pharos FX Plus molecular imager using an excitation wavelength of 532 nm. Scans were quantified with Image J (NIH). coverslips 4% paraformaldehyde Triton X-100/PBS (pH 7.2) containing 1% BSA. Specimens were incubated with anti-PP2A B56 α (1:200) or anti-PP2AC (1:20) and anti-RyR2 (1:1,000) primary antibodies followed by fluorophore-conjugated, highly cross-adsorbed IgGs (Alexa Fluor-488 and Alexa Fluor-633; 1:200, Invitrogen Corp., CA). Images were acquired using Olympus Fluoview 1000 confocal system equipped with 60X1.4 N.A. oil objective. Statistical Analysis. Results are presented as Mean±SEM. Statistical significance was evaluated either by Student's t -test for paired observations or by One Way ANOVA as appropriate. P<0.05 was considered significant. was undetectable in experimental conditions comparable to the conditions used in Ca cycling studies for both control and miR-1 groups. Similar results were obtained in 5 independent experiments.