Pharmacological inhibition of CDC25 dual-specificity phosphatases prevents the induction of hypertrophy in cardiomyocytes

Katrina Bicknell, Carmen Coxon, Gavin Brooks
University of Reading, Reading, United Kingdom

Cardiac hypertrophy is an adaptive growth process whereby the mature heart responds to increased load and/or injury. However, prolonged periods of chronic stress on the heart can result in excessive hypertrophic growth that can adversely affect cardiac function leading to heart failure. An understanding of the cellular mechanisms involved in the hypertrophic growth response might identify molecules that can be targeted to prevent detrimental hypertrophic growth. Induction of hypertrophy in cardiomyocytes involves a partial reactivation the cell cycle machinery with the activities of G1/S-acting cyclin:cyclin-dependent kinase (CDK) complexes increasing transiently (Li et al., 1998). Since the activities of these cyclin:CDKs, at least in part, is regulated by CDC25 phosphatases, the present study investigates the role of CDC25 in modulating cardiomyocyte hypertrophy. We have investigated the regulation of the protein expressions of CDC25 phosphatases during the induction of cardiomyocyte hypertrophy using an established in vitro model that employs cultured neonatal cardiomyocytes isolated from 2-3 day old Wistar rat hearts. We also have used cultured adult cardiomyocytes, isolated from the hearts of 200-250g male Wistar rats, to investigate the effect that CDC25 inhibitors, 2-(2-mercapto-1-hydroxyethyl)-3-methyl-1,4-naphthoquinone (NSC672121; Tamura et al., 2000) or 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC95397; Lazo et al., 2002), have upon serum-induced hypertrophy. All animals used in this study were euthanised using cervical dislocation in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Abrogation of cardiac hypertrophy was demonstrated by the inhibition of the induction of atrial natriuretic peptide (ANP) mRNA expression, a marker of cardiac hypertrophy. All results are presented as means ± standard error of the mean. We have shown that hypertrophic stimulation of neonatal cardiomyocytes with serum (20%), phenylephrine (100μM) or endothelin-1 (10nM) significantly increased G1/S phase-acting CDC25A expression but had no effect on expression of G2/M phase-acting CDC25C; G2 phase-acting CDC25B expression was increased only by serum and phenylephrine. Co-treatment of adult cardiomyocytes with 20% serum and 0.5μM or 1μM NSC672121 significantly reduced induction of ANP expression to 1.63±0.15 fold or 1.51±0.33 fold, respectively, compared to ANP expression in cultures treated with serum alone (2.47±0.04 fold) or serum starved cardiomyocytes (adjusted to 1.00 fold; n=3). Similar experiments using the more potent inhibitor NSC95397 (0.25μM) also demonstrated that serum-stimulated induction of ANP expression in adult cardiomyocytes was reduced significantly by inhibiting CDC25 phosphatases. Taken together, increased expression of CDC25 in cardiomyocytes following exposure to hypertrophic stimuli and abrogation of the induction of hypertrophy in the presence of CDC25 inhibitors suggests a specific role for CDC25 in hypertrophic growth. Thus, this study has shown that CDC25 phosphatases might represent a novel therapeutic target for controlling detrimental hypertrophic growth.

Lazo et al., 2002. Mol. Pharmacol 61: 720-728
Li et al., 1998 Am J Physiol. 275:H814-22.
Tamura et al., 2000. Cancer Res 60:1317-1325