Abstract 13216: NFATc3-dependent miR26a Regulation Underlies TRPC3-dependent Fibroblast Activation in AF
Background: Cardiac fibroblast (FB) proliferation and differentiation into activated myofibroblasts contributes to AF-promoting remodeling. MicroRNAs (miRs) post-transcriptionally regulate protein expression.We previously reported Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channel-dependent FB activation in a canine AF model. Here, we examined the regulatory mechanisms controlling TRPC3 and FB function.
Methods: Bioinformatic analysis predicted miR26 targeting of TRPC3. HEK293 cells were transfected with luciferase vectors containing the miR26a target sequence of TRPC3. LA FBs isolated from AF (600 beats/min atrial pacing, 1 week, n=9) and control dogs (CTL, n=28) were used for qPCR, Western blotting, immunostaining and proliferation analysis (FACscan).
Results: AF increased FB TRPC3 protein expression by 64%* (*p<0.05) and reduced FB expression of miR26a by 65%*. Knockdown of endogenous miR26a by antisense oligos (AMO26a) increased luciferase signals by 46%*, indicating TRPC3 translation-inhibition by miR26a. In cultured CTL FBs, miR26a knockdown to mimic AF-induced miR26a downregulation increased TRPC3 protein, G2/M-cell percentage (index of mitosis) and FB count by 53%*, 24%* and 37%*, respectively, showing that miR26a regulates TRPC3 protein and FB proliferation. MiR26 has Nuclear Factor of Activated T-cell (NFAT) binding sites in the 5’ promoter region. NFAT showed nuclear translocation in AF FBs based on: 1. Immunostaining: nuclear/cytosolic ratio increased by 135%*; 2. Nuclear-fraction NFATc3 protein expression (relative to nuclear marker lamin A/C) increased in AF by 630%* whereas cytosolic expression (relative to cytosolic marker HSP70) decreased by 55%*. In cultured CTL FBs, treatment with a selective NFATc3/c4 blocker, INCA6 (2.5 µM), increased miR26a by 31%* and decreased TRPC3 protein by 28%*, suggesting that in AF NFAT activation suppresses miR26a transcription, antagonizing miR26-inhibition of TRPC3 protein translation.
Conclusion: MiR26a suppresses TRPC3 protein expression and FB proliferation and is negatively regulated by NFATc3. AF increases NFATc3 nuclear translocation; thus, NFATc3-dependent miR26a regulation underlies TRPC3-dependent fibroblast activation in AF.
- © 2012 by American Heart Association, Inc.