Abstract
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 Ca2+ imaging revealed that in electrically stimulated myocytes overexpressing miR-129, Ca2+ transient amplitudes were significantly reduced compared to controls. In contrast, sarcoplasmic reticulum (SR) Ca2+ contents assessed by rapid application of caffeine remained unchanged. Furthermore, kinetic analyses of Ca2+ transient decays suggested miR-129 overexpression does not affect Na+/Ca2+ exchanger or SR Ca2+ ATPase function. Voltage-clamp experiments showed no difference in Ca2+ currents in miR-129 overexpressing and control cells. Additionally, permeabilized myocytes demonstrated no change in Ca2+ spark frequency nor amplitude indicating no effect from miR-129 overexpression on SR Ca2+ release channel Ryanodine Receptor (RyR2) function. Detailed analysis of Ca2+ transient rising phase revealed significant time-to-peak prolongation accompanied by reduction in Ca2+ 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 Ca2+ channels and RyR2s located in junctional SR. Efficient coupling between L-type Ca2+ 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 Ca2+ release in HF by downregulation of JPH4 thus altering geometry of the dyadic cleft.
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