Abstract 14803: KChIP2 Leads to Changes in Cardiac Gene Expression Through Transcriptional Control of miR-34s
Introduction: Cardiac ion channels and their accessory subunits are critical in maintaining proper cardiac electrical activity. Importantly, the K+ channel interacting protein 2 (KChIP2) is emerging as a player in multiple modes of cardiac action potential regulation. Initially identified as an ancillary subunit for Kv4 channels, KChIP2 has since shown more extensive effects including influences on transcript and protein expression of multiple ion channels. Interestingly, a disconnect observed between transcripts and proteins patterns suggest a mechanism compatible with altered miRNA activity. Considering other members of the KChIP family behave as transcriptional repressors, we hypothesized that KChIP2 regulates discrete microRNAs which in turn regulate cardiac excitability.
Methods and Results: A miRNA microarray study was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2 by siRNA. This led to the identification of the miR-34 family as potential transcriptional targets of KChIP2. This regulation was confirmed by quantitative PCR, showing a reduction in miR-34a/b/c when over-expressing KChIP2 and an increase following silencing. To assess whether KChIP2 modulated the transcriptional activity of these miRNAs directly, reporter assays were conducted on the cloned promoter of miR-34b/c inserted into the pGL4 luciferase vector. Expression of this vector with KChIP2 confirmed its repressive behavior on the miR-34b/c promoter. Furthermore, chromatin immunoprecipitation followed by PCR of putative binding sites confirmed direct binding of KChIP2 to the miR-34b/c promoter. Since modified expression of KChIP2 can lead to changes in several ion channel subunits, we investigated whether this was the consequence of KChIP2 regulation via miR-34. Therefore, a miR-34b precursor was expressed in NRVM which reduced the transcript levels of Nav1.5. Conversely, transcript levels for Kv4.3 were maintained but with reduced protein expression.
Conclusion: Taken together, these observations describe a novel mechanism where KChIP2 regulates a host of cardiac genes through transcriptional control of miRNAs, potentially explaining electrical remodeling observed in disease states where KChIP2 is reduced.
- © 2013 by American Heart Association, Inc.