K_V1.5 channel down‐regulation in pulmonary hypertension is nothing short of MiR‐1‐aculous!

Pulmonary hypertension (PH), defined as a pulmonary artery (PA) pressure of ≥25 mmHg, can arise idiopathically, as a consequence of inheritance, or secondary to other conditions. The most severe form of PH, pulmonary arterial hypertension (PAH; Group 1 PH), is characterized by an increased pulmonary vascular resistance associated with PA vasoconstriction and severe pulmonary vascular remodelling; this leads to right heart failure, with a grave prognosis. Although an integrated understanding of PAH pathogenesis remains elusive, abundant evidence indicates that down‐regulation of several types of K⁺ channels in PA smooth muscle cells (PASMC), causing membrane depolarization and a consequent increase in intracellular [Ca²⁺], may play an important causative role. In particular, the expression of K_V1.5 (KCNA5) the most important voltage‐gated K⁺ current (VGKC) in PASMC, is diminished in various animal models and clinical manifestations of PH, including PAH.

The mechanisms responsible for K_V1.5 down‐regulation in PAH are unknown. However, in the current issue of The Journal of Physiology, a paper from Francisco Perez‐Vizcaino and Angel Cogolludo's laboratory at the Universidad Complutense de Madrid (Mondejar‐Parreño et al. 2019) presents novel evidence that the decreased expression of PASMC K_V1.5 is caused by increased levels of microRNA‐1 (miR‐1) in a model of PH which recapitulates many of the features of human PAH.

MicroRNAs (miRs) are small (∼22 nucleotides) single‐stranded non‐coding RNAs that inhibit gene expression post‐transcriptionally. They bind to microRNA responsive elements (MREs) in the 3’UTR of their target mRNAs, causing them to be degraded and/or inhibiting their translation into protein. MicroRNAs can be transcribed like any gene: they may contain promoters and have tightly regulated transcription. They can also arise from processing of intronic or exonic sequences of coding and non‐coding genes. The base pairing between microRNAs and their targets is imperfect; thus one single microRNA can target hundreds of different mRNAs, thereby regulating an entire biological pathway. MicroRNAs are involved in virtually all biological processes from development to ageing, inflammation and cardiac development. MicroRNAs can also be applied exogenously as pre‐microRNAs or inhibited using anti‐microRNAs (antagomiRs).

The possibility that miR‐1 might be affecting K_V1.5 expression emerged when Mondejar‐Parreño et al. carried out an in silico analysis identifying KCNA5 as a potential miR‐1 target. They then showed that the expression in COS‐7 cells of a luciferase reporter vector incorporating the 3’UTR of KCNA5 was diminished by co‐transfection with miR‐1, but not with (negative) scrambled miR. It is worth pointing out, however, that they used the 3’UTR from human KCNA5 in this construct (and also employed human mimic miR‐1 in the over‐expression experiments described below), whereas they should have used a rat miR‐1 mimic and demonstrated direct targeting of the rat Kcna5 3’UTR. Whilst the sequences of human and rat miR‐1 differ by only one nucleotide, which is outside of the ‘seed‐region’ (the primary determinant of target specificity), changes outside of this region may affect targeting (Broughton et al. 2016). Although it is very likely that the human mimic miR‐1 was binding to the rat Kcna5 3’UTR given the evidence of functional effect presented in the rest of the manuscript, ideally they would have pinpointed the predicted binding site of miR‐1 by showing that site‐directed mutagenesis of this site prevents miR‐1 from down‐regulating the reporter; this was not done. Thus, the exact mechanism by which the miR‐1 mimic they used was acting on Kcna5 was not established.

They compared the VGKC and K_V1.5 expression in PASMC from control rats and those in which PH was induced by combined treatment with chronic hypoxia and the VEGF antagonist Su5461. The VGKC, which was largely suppressed by the K_V1.5‐selective antagonist DPO‐1, was profoundly reduced in the PH animals compared to controls. Moreover, protein expression of K_V1.5 was diminished in lung homogenates from the PH rats compared to controls, whereas miR‐1 expression was increased fourfold. In order to determine whether these events were causally linked, they used an innovative method of transfecting miR‐1 into isolated PA, which involved briefly digesting the tissues with collagenase and elastase prior to incubating them with miR‐1 (and/or scrambled miR or antagomiR‐1) in the presence of Lipofectamine, at the same time applying a reverse permeabilization protocol in which extracellular Ca²⁺ was removed and then gradually replaced.

Using this approach, they found that, compared to PASMC treated with scrambled miR, cells from arteries transfected with miR‐1 showed decreased K_V1.5. protein levels, were depolarized, and exhibited a much smaller VGKC. Moreover, transfection with antagomiR‐1 greatly increased the amplitude of the VGKC and the protein expression of K_V1.5 in PA which had been exposed to hypoxia and Su5416 for 48 h, whilst it did not affect either the K⁺ current or channel expression in control normoxic PA.

In summary, the results demonstrate that pulmonary miR‐1 expression is upregulated in an important animal model of PAH, and that this causes attenuation of K_V1.5 expression in PASMC. However, both increases (Sarrion et al. 2015) and decreases (Wei et al. 2013) in miR‐1 expression have been reported in patients with PH, and could reflect heterogeneous consequences associated with different types of the disease. Indeed, a pathway involving HIF1α and ET‐1, both of which contribute importantly to PH pathogenesis, has been shown to suppress K_V1.5 channel expression in the chronic hypoxia rat model of PH (Whitman et al. 2008), and since there is evidence that miR‐1 can reduce levels of HIF1α and ET‐1 in some types of cells (Xu et al. 2017), it is possible that a decrease in miR‐1 levels could also down‐regulate K_V1.5 channels in some types of PH, as the authors discuss. Therefore, a better understanding of the precise role(s) of miR‐1 in the regulation of these channels during PH will require additional studies using cells from patients with different types of PH. Hopefully this pioneering study will engender an increased effort among workers in this field to further elucidate the involvement of miR‐1 and other microRNAs in the pathogenesis of this perplexing disease.

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Competing interests

The authors declare that they have no conflicts of interest relating to this work.