Myocardial MiR-30 downregulation triggered by doxorubicin drives alterations in β-adrenergic signaling and enhances apoptosis

Abstract

The use of anthracyclines such as doxorubicin (DOX) has improved outcome in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF) but our understanding of molecular mechanisms underlying this and essential for development of cardioprotective strategies remains largely obscure. As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in DOX-induced HF and the alteration of cellular processes downstream. Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes or via chronic exposure in vivo, and compared with miRNA profiles from remodeled hearts following myocardial infarction. The miR-30 family was downregulated in all three models. We describe here that miR-30 act regulating the β-adrenergic pathway, where preferential β1- and β2-adrenoceptor (β1AR and β2AR) direct inhibition is combined with Giα-2 targeting for fine-tuning. Importantly, we show that miR-30 also target the pro-apoptotic gene BNIP3L/NIX. In aggregate, we demonstrate that high miR-30 levels are protective against DOX toxicity and correlate this in turn with lower reactive oxygen species generation. In addition, we identify GATA-6 as a mediator of DOX-associated reductions in miR-30 expression. In conclusion, we describe that DOX causes acute and sustained miR-30 downregulation in cardiomyocytes via GATA-6. miR-30 overexpression protects cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective and anti-tumorigenic strategy for anthracyclines.

Figure 1

miRNA expression dysregulation occurs upon acute and sustained DOX treatment. (a) Experimental design of the models of study included in the miRNA profiling. DOX-induced HF in vivo model was generated by six i.p. DOX injections (2.5 mg/kg) spread over a fortnight. MI was induced by proximal LAD coronary artery ligation. For acute DOX treatment, cultured viable ARVCM were treated with 1 μmol/l for 6 h. For all three models, RNA was isolated specifically from viable ARVCM. (b) Venn diagram showing significantly altered miRNA expression (>1.5-folds, P<0.05) detected for the profiled models of cardiac injury (n=3 per group). The three downregulated members of the miR-30 family are highlighted in yellow. Several other miRNAs have previously been linked to heart disease when dysregulated in the same direction as we observed and appear in bold writing. (c) miRNAs that presented reduced expression levels in at least two experimental models. rno, rattus norvegicus.

Figure 2

miR-30 target prediction and validation of direct 3′UTR regulation by 3'UTR luciferase assay. (a) Pathway enrichment analysis of the predicted miR-30 targets by TargetScan; performed using Database for annotation, visualization and integrated discovery. Three types of cardiomyopathy are part of the top 10 most significantly enriched pathways. (b) Representation of miR30e-mRNA annealing for four of the target genes predicted by TargetScan (β₁AR, β₂AR, BNIP3L and G_iα-2). miRNA seed sequence is highlighted in red. (c) Relative luciferase activity measured on H9c2 cultures, after 24 h of co-transfection with either pre-30e or pre-NC (100 nmol/l) and the correspondent (wild type (WT) or mutant (mut)) 3′UTR luciferase reporter vector for each of the potential targets. The non-target programed cell death protein 4 (PDCD4) was included as negative control. Averaged (±S.E.M.) values are the result of at least three independent transfections (ns, not significant; t-test)

Figure 3

Experimental confirmation of β1AR, β2AR, BNIP3L and Giα-2 as miR-30 targets. (a) RT-qPCR shows relative target expression levels when overexpressing miR-30. All values are normalized to U6 levels and presented as ratios to the pre-NC-transfected controls (20 nmol/l). Three biological replicates were performed on H9c2 cultures. (b) Relative protein levels of BNIP3L and G_iα_-2 were measured by western blot; band densitometry was quantified by ImageJ and normalized to tubulin. (c) Schematic view of the sponge vector designed for miR-30 family inhibition (pEGFP-sp30), using the pEGFP-C1 plasmid as backbone. EGFP expression in transfected cultures was detected by microscopy. (d) Relative target mRNA levels measured by RT-qPCR and protein levels (e) upon transfection with pEGFP-sp30 for miR-30 family inhibition, or the control vector pEGFP-C1. Data are averages of three biological replicates performed on H9c2 cardiac cells. (f) Target expression levels measured by RT-qPCR following DOX treatment (1 μmol/l, 18 h), alone or in combination with miR-30e overexpression (pre-30e transfection). Three independent replicates were performed on H9c2 cells, being presented as averaged ratio to control ±S.E.M. (g) RT-qPCR data showing expression levels of the predicted miR-30 targets in vivo in DOX-treated hearts (15 mg/kg cumulative dose, n=5 rats per group). Student's t-test performed for statistical analysis. Error bars represent±S.E.M.

Figure 4

miR-30 effects on cAMP accumulation and contractility. (a) cAMP accumulation data expressed as % of the respective control samples. Values are the mean±S.E.M. of three independent experiments. The diagram on the right shows the contribution to cAMP production of the three miR-30 targets involved in the β-adrenergic pathway. (b) Baseline % of contraction amplitude of paced ARVCM transfected with either pre-30e or pre-NC for 48 h (100 nmol/l), measured using IonOptix (n=8–10 cells from 6 preparations; t-test; ns, not significant). (c) Contractile responses of isoproterenol-stimulated transfected ARVCM, recorded using IonOptix (n=6 cells from 6 preparations, F-test)

Figure 5

High miR-30 levels are protective against DOX insult and reduce ROS generation. (a) Left graph: caspase activity in cardiac cultures treated with DOX alone (1 μmol/l, 18 h) and in combination with miR-30e overexpression through pre-30e mimic transfection (20 nmol/l). Right graph: relative caspase activity upon miR-30 family inhibition using the pEGFP-sp30 sponge vector. (b) Bax and Bcl-2 protein levels measured by western blot analysis. Cells were treated with DOX (1 μmol/l, 6 h and 18 h) and also transfected with pre-30e mimics (20 nmol/l) in combination with DOX (1 μmol/l, 18 h). Band densitometry was measured using Image J. All values are normalized to Tubulin and expressed as Bax/Bcl-2 ratio. (c) ROS generation of transfected cultures for miR-30 overexpression or inhibition, measured by DCFDA fluorescence intensity. All assays (a, b, c) were performed on H9c2 cells and results are average of independent triplicates ±S.E.M. (t-test; ns, not significant)

Figure 6

GATA-6 mediates miR-30 downregulation triggered by DOX. (a) RT-qPCR data showing GATA-6 and miR-30e expression levels in response to DOX time-course treatment (1 μmol/l). (b) Relative levels of primary and mature miR-30d and miR-30e, upon GATA-6 silencing detected by RT-qPCR. (c) RT-qPCR data for miR-30 target mRNA levels in response to GATA-6 silencing (25 nmol/l, 72 h) and after co-transfection with compensatory LNA miR-30 family inhibitors (100 nmol/l, 72 h). (d) Relative caspase activity in response to GATA-6 silencing, ±DOX treatment (1 μmol/l, 18 h). All values (a, b, c, d) were obtained performing biological triplicates on H9c2 cells and are presented as averaged ratio to the respective control±S.E.M. All RT-qPCR results were normalized to the correspondent U6 Ct value (ns, not significant; t-test)

Figure 7

Proposed model: importance of the miR-30 regulatory effects in cardiomyocytes following DOX exposure. Upon DOX treatment, there seems to be an acute increase of GATA-6 expression in cardiomyocytes. GATA-6 is able to repress miR-30 family expression, resulting in DOX-induced miR-30 downregulation. In turn, the expression levels of miR-30 targets are upregulated. Three of the miR-30 target genes are involved in the complex catecholamine/adrenergic pathway. A correct equilibrium of members is crucial in this signaling cascade, as AC-cAMP-PKA activation promotes cardiomyocyte contraction, whereas Gi impairs contraction but protects against apoptosis via Phosphoinositide 3-kinase/Protein kinase B. In addition, miR-30 represses BNIP3L, which is involved in mitochondrial death. miR-30 expression seems to be essential in regulating mitochondrial apoptotic pathways and it is able to partially counteract DOX-induced toxicity. Solid lines: previously described mechanisms, dashed lines: novel mechanisms