miR-1 overexpression enhances Ca(2+) release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha 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+) cycling in rat ventricular myocytes using methods of electrophysiology, Ca(2+) 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+) current, flattening of Ca(2+) transients voltage dependence, and enhanced frequency of spontaneous Ca(2+) sparks while reducing the sarcoplasmic reticulum Ca(2+) content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca(2+), 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+)/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 B56alpha 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.
Terentyev
Dmitry
D
Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210, USA. Dmitry.Terentyev@osumc.edu
Belevych
Andriy E
AE
Terentyeva
Radmila
R
Martin
Mickey M
MM
Malana
Geraldine E
GE
Kuhn
Donald E
DE
Abdellatif
Maha
M
Feldman
David S
DS
Elton
Terry S
TS
Györke
Sandor
S
D000256:Adenoviridae / Q000235:genetics; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D001596:Benzylamines / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D005822:Genetic Vectors; D007545:Isoproterenol / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D009200:Myocardial Contraction / Q000187:drug effects / Q000235:genetics; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic; D000096926:Benzenesulfonamides
D000318:Adrenergic beta-Agonists; D001596:Benzylamines; D020746:Calcium Channels, L-Type; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D007545:Isoproterenol; D035683:MicroRNAs; D047428:Protein Kinase Inhibitors; D054648:Protein Phosphatase 2; D019837:Ryanodine Receptor Calcium Release Channel; D013449:Sulfonamides; C517731:Mirn1 microRNA, mouse; C072105:KN 93; D000096926:Benzenesulfonamides; C513662:B56alpha protein, rat