Excitation-Contraction Coupling II

2801-Pos Board B571 Increased miR-129 in Heart Failure Leads to Diminished Calcium Release by Downregulation of Junctophilin 4 Jennifer A. Rochira1, Karim Roder1, Weiyan Li1, Radmila Terentyeva2, Yukiko Kunitomo3, Terry S. Elton4, Dmitry Terentyev1. RI Hospital/Brown University, Providence, RI, USA, RI Hospital, Providence, RI, USA, Brown University, Providence, RI, USA, Ohio State University, Columbus, OH, USA. MicroRNAs (miRNAs) are small nonprotein-coding RNA molecules that target mRNAs for translational repression or degradation. Recent studies demonstrated that miRNA expression levels are substantially altered in heart failure (HF) and therefore may play a role in the functional abnormalities observed in diseased hearts. Since miR-129 expression is augmented in HF, we hypothesized that HF-observed changes in Excitation-Contraction Coupling result, in part, from miR-129-mediated downregulation of specific calcium handling proteins. Confocal Ca2þ imaging revealed that in electrically stimulated myocytes overexpressing miR-129, Ca2þ transient amplitudes were significantly reduced compared to controls. In contrast, sarcoplasmic reticulum (SR) Ca2þ contents assessed by rapid application of caffeine remained unchanged. Furthermore, kinetic analyses of Ca2þ transient decays suggested miR-129 overexpression does not affect Naþ/Ca2þ exchanger or SR Ca2þ ATPase function. Voltage-clamp experiments showed no difference in Ca2þ currents in miR-129 overexpressing and control cells. Additionally, permeabilized myocytes demonstrated no change in Ca2þ spark frequency nor amplitude indicating no effect from miR-129 overexpression on SR Ca2þ release channel Ryanodine Receptor (RyR2) function. Detailed analysis of Ca2þ transient rising phase revealed significant time-to-peak prolongation accompanied by reduction in Ca2þ release synchronization in miR-129 overexpressing myocytes compared with controls. Overall, these data imply that miR-129 overexpression reduces the fidelity of coupling between plasmalemmal Ca2þ channels and RyR2s located in junctional SR. Efficient coupling between L-type Ca2þ channels and RyR2 is facilitated by junctophilins (JPH), proteins tethering T-tubules to SR forming junctional membrane complexes keeping channels at precise distances. Western blot analyses demonstrated that miR-129 overexpression significantly reduced levels of JPH4, a putative target of miR-129. Therefore, our results suggest that increased levels of miR-129 may underlie diminished Ca2þ release in HF by downregulation of JPH4 thus altering geometry of the dyadic cleft.