Abstract
Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.
Highlights
Mineralocorticoids, in particular aldosterone, are associated with the development of cardiac hypertrophy [1] and heart failure [2]
It has been shown that cardiac T-type voltage-gated Ca2+ channel expression and activity are upregulated by aldosterone in neonatal rat cardiomyocytes [10,11,15]
We found here that microRNA 204 (miR-204) is the main microRNA upregulated by aldosterone in cardiomyocytes
Summary
Mineralocorticoids, in particular aldosterone, are associated with the development of cardiac hypertrophy [1] and heart failure [2]. It has been shown that cardiac T-type voltage-gated Ca2+ channel expression and activity are upregulated by aldosterone in neonatal rat cardiomyocytes [10,11,15]. In order to confirm that miR-204 expression is necessary to allow aldosterone to stimulate L-type and T-type Ca2+ channel upregulation, we used an antisense RNA to target miR-204 (named antagomir-204). Neonatal rat cardiomyocytes were transfected with antagomir-204 to block miR-204 expression induced by the hormone, while a control antisense RNA was used in control cells. Because miRNAs promote the downregulation of their specific targets [23], we hypothesized that Binecoarudseer mtoiRpNroAmsopteroinmcoreteastehde Td-ochwannrneegluelaxtpiorenssoifonthuepirosnpaelcdifoicsttearrogneetsst[i2m3]u, lwateiohny, pmoitRhNesAiz2e0d4 tmhautsitndoorwdnerretgouplartoemthoeteexinpcrreesassioedn oTf-cohnaenonresleevxeprraelsnseiognatuivpeotnraanldscorsiptetrioonnaelsrteimguulalattoirosno, fmTi-RchNaAnn20e4ls mthuasttadroewalnrreeagduylaetxeptrheesseexdprienssreiosntinogf ocneellsoransedvtehrearlenfeograetpivreevtreanntscchriapntnioenlaelxrpergeuslsaiotonr.sRoEf TST-c-hNaRnSnFeliss tahaktnaorwe naltrreaandsycreixpptiroenssaeldreipnrreessstoinr gthcaetllbslaonckdsththeeretfroarnescprriepvteionnt cahnadnenxepl reexspsrioesnsioofnt.hReECSaTV-N3.R2 STF-tiyspae known transcriptional repressor that blocks the transcription and expression of the CaV3.2 T-type channel in cardiac cells [29]. Luciferase siRNA (sense strand, 5 -CUUACGCUGAGUACUUCGATT-3 ; antisense strand, 5 -UCGAAGUACUCAGCGUAAGTT-3 ; where lower cases indicate DNA) was purchased from Gene Design Inc. (Osaka, Japan)
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