DYRK1A is a novel negative regulator of cardiomyocyte hypertrophy

Abstract

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors.

FIGURE 1.

DYRK1A is induced by hypertrophic agents in vitro. A, treatment of cardiomyocytes with PE (50 μmol/liter) and endothelin-1 (ET-1, 100 nmol/liter) for 24 h induces DYRK1A protein expression. B, PE as well as ET-1 lead to a significant induction of DYRK1A (PE: 1.6 ± 0.05-fold, ET-1: 1.4 ± 0.03-fold, p < 0.001, n = 6).

FIGURE 2.

DYRK1A inhibits cardiomyocyte hypertrophy induced by phenylephrine. A, representative images of β-galactosidase (25 moi) and DYRK1A (25 moi)-overexpressing cardiomyocytes stained with an α-actinin antibody reveal the resistance of DYRK1A-overexpressing NRVMs to PE-induced hypertrophy. DAPI is used as counterstain for nuclei. B, while stimulation with PE (100 μmol/liter) for 24 h leads to a 1.5 ± 0.04-fold increase in cardiomyocyte surface area in control cells (p < 0.001), AdDYRK1A treatment abrogates cardiomyocyte hypertrophy induced by PE (p < 0.001, 50 cells per condition, n = 3). C, AdDYRK1A (25 moi) reverses the induction of ANF- (−67.8%, p < 0.01, n = 6) and D, BNP-mRNA (−75.8%, p < 0.001, n = 6) caused by PE (100 μmol/liter, 24 h). E, catalytically inactive point mutant of DYRK1A (AdK188R) causes cellular hypertrophy (1.3 ± 0.04-fold, p < 0.001) and fails to inhibit PE-induced hypertrophy (100 μmol/liter, 24 h). The cell surface area of PE-stimulated cardiomyocytes overexpressing DYRK1A-K188R did not differ from PE-treated cells overexpressing the control virus (50 cells per condition, n = 3). Scale bars, 10 μm.

FIGURE 3.

DYRK1A reverses cardiomyocyte hypertrophy induced by calcineurin. A, representative α-actinin immunofluorescence images of cardiomyocytes overexpressing a constitutively active mutant of CnA and DYRK1A. B, while the overexpression of CnA causes a significant increase in cardiomyocyte surface area (1.6 ± 0.04-fold, p < 0.001, 100 cells per condition, n = 3), DYRK1A inhibits the CnA-induced increase in cell surface area by 78% (p < 0.001). The antihypertrophic effect of DYRK1A is accompanied by a reduction of CnA-induced (C) ANF-mRNA by −60% (p < 0.05, n = 3) and (D) BNP-mRNA by −56% (p < 0.05, n = 3). Scale bars, 10 μm.

FIGURE 4.

Knockdown of DYRK1A causes cardiomyocyte hypertrophy. A, evaluation of the knockdown efficiency of three adenovirally encoded synthetic miRNAs (AdmiR-544, -1331, -1732; 25 moi each) against DYRK1A reveals (B) AdmiR-1732 as the most potent miRNA with −70.1% down-regulation of DYRK1A protein expression (n = 2). C, likewise, this synthetic miRNA reduces DYRK1A mRNA levels by −76.9% (p < 0.01, n = 3). D, representative cardiomyocytes stained with an α-actinin antibody after adenovirally delivered miRNA against DYRK1A (10 moi). The adenovirally delivered knockdown construct carries a GFP cassette. E, only double-labeled cells were measured, revealing a significant increase in cell surface area (1.8 ± 0.05-fold, p < 0.001, 75 cells per condition, n = 3) in response to DYRK1A down-regulation. F, hypertrophic phenotype is accompanied by a 9.8 ± 2.8-fold induction of ANF-mRNA (p < 0.05, n = 8). Scale bars, 10 μm.

FIGURE 5.

Pharmacological inhibition of DYRK1A results in cardiomyocyte hypertrophy. A, representative α-actinin immunofluorescence images of NRVMs treated with vehicle, DYRK1A inhibitor harmine (1 μmol/liter) or PE (50 μmol/liter) for 24 h. B, treatment of NRVMs with harmine causes an increased cell surface area (1.6 ± 0.04-fold, p < 0.001, 100 cells per condition, n = 3). C, hypertrophic phenotype is accompanied by an induction of ANF-mRNA up to 2.2 ± 0.2-fold (p < 0.05, n = 3). Scale bars, 10 μm.

FIGURE 6.

DYRK1A attenuates NFAT-activity in cardiomyocytes. A, AdDYRK1A reduces calcineurin (AdCnA)-induced NFAT luciferase reporter activity dose dependently up to −50.1% (n = 3). B, conversely, AdmiR-DYRK1A increases reporter activity up to 4.3 ± 0.5-fold and exaggerates the calcineurin effect up to 2-fold (n = 12–18). C, likewise, the inhibition of DYRK1A by harmine (100 nmol/liter) exacerbates the effect of calcineurin up to 1.5-fold (p < 0.01, n = 9). D, expression of RCAN 1–4, a direct target gene of NFAT, is found markedly up-regulated in harmine-treated cardiomyocytes (1 μmol/liter, 24 h, n = 3).

FIGURE 7.

Proposed role of DYRK1A in cardiomyocyte hypertrophy. DYRK1A directly phosphorylates NFAT thereby mediating its nuclear export which leads to a reduced transcriptional activity of prohypertrophic target genes.