The A-769662 compound is a specific activator of the AMP-activated protein kinase (AMPK). Several studies have shown that unspecific AMPK activators, like resveratrol and metformin, inhibit cardiac hypertrophy by preventing the increase in protein synthesis. This is accompanied by the modification of the phosphorylation state of two proteins involved in protein translation, namely p70 ribosomal S6 protein kinase (p70S6K) and elongation factor 2 (eEF2). The aim of this work was to evaluate the putative therapeutic effect of A-769662 for the treatment of cardiac hypertrophy.Methods
Cultured neonatal rat cardiomyocytes were stimulated with phenylephrine (PE, 10μM) in the presence or not of A-769662 (100 μM) during 24h. Cardiomyocyte hypertrophy was visualized by immunostaining of α-actinin and by QPCR for ANP and BNP. Protein synthesis was measured by incorporation of radioactive methionine into proteins. Phosphorylation of AMPK, p70S6K, eEF2 and ERK was evaluated by immunoblot. Translocation of NFAT was studied by immunostaining.Results
A-769662 was able to chronically stimulate AMPK signalling in cardiomyocytes. A-769662 treatment prevented the PE-induced cardiomyocyte hypertrophy. PE increased the mRNA level of both BNP and ANP and this was inhibited by A-769662 (Ctrl: 1 ± 0.03; PE: 2.49 ± 0.7; PE + A: 0.97 ± 0.21 in relative values for BNP and Ctrl: 1.07 ± 0.07; PE: 2.17 ± 0.27; PE + A: 0.82 ± 0.35 for ANP). This is associated with a decrease in PE-induced stimulation of protein synthesis (Ctrl: 4117 ± 621; A:4873 ± 204; PE: 7463 ± 854; PE + A: 4435 ± 777 incorporated dpm/dish, p < 0.05). A-769662 blocked the PE-mediated increase in p70S6K phosphorylation (Ctrl: 0.96 ± 0.11; A: 0.53 ± 0.10; PE: 2.54 ± 0.23; PE + A: 1.3 ± 0.20 arbitrary unit, p < 0.05). Similarly, A-769662 prevented the decrease in eEF2 phosphorylation induced by PE (Ctrl: 1 ± 0.07; A: 1.57 ± 0.09; PE: 0.49 ± 0.07; PE + A: 1.17 ± 0.11, arbitrary unit, p < 0.05). Moreover, PE promoted the nuclear translocation of NFAT, a transcription factor located downstream calcineurin and essential in the development of cardiac hypertrophy. This PE-induced translocation was reversed by the A-769662 treatment (Ctrl: 24 ± 2; A: 27 ± 2; PE: 85 ± 2; PE + A: 28 ± 1 % of NFAT-positive nuclei). Finally, the A-769662 compound blocked the increase in ERK phosphorylation caused by PE treatment (Ctrl: 1.00 ± 0.11; A: 0.71 ± 0.06; PE: 1.31 ± 0.08; PE + A: 0.61 ± 0.13 arbitrary unit, p < 0.05).Conclusions
Activation of AMPK by A-769662 appears to be a potential therapeutic approach to treat cardiac hypertrophy by inhibiting protein synthesis and transcription factor signalling pathways.