Neuronal NO synthase mediates plenylephrine induced cardiomyocyte hypertrophy through facilitation of NFAT-dependent transcriptional activity

Abstract

Neuronal nitric oxide synthase (NOS1) has been consistently shown to be the predominant isoform of NOS and/or NOS-derived NO that may be involved in the myocardial remodeling including cardiac hypertrophy. However, the direct functional contribution of NOS1 in this process remains to be elucidated. Therefore, in the present study, we attempted to use silent RNA and adenovirus mediated silencing or overexpression to investigate the role of NOS1 and the associated molecular signaling mechanisms during OKphenylephrine (PE)-induced cardiac hypertrophy growth in neonatal rat ventricular cardiomyocytes (NRVMs). We found that the expression of NOS1 was enhanced in PE-induced hypertrophic cardiomyocytes. Moreover, LVNIO treatment, a selective NOS1 inhibitor, significantly decreased PE-induced NRVMs hypertrophy and [³H]-leucine incorporation. We demonstrated that NOS1 gene silencing attenuated both the increased size and the transcriptional activity of the hypertrophic marker atrial natriuretic factor (ANF) induced by PE stimulation. Further investigation suggested that deficiency of NOS1-induced diminished NRVMS hypertrophy resulted in decreased calcineurin protein expression and activity (assessed by measuring the transcriptional activity of NFAT) and, an increased activity of the anti-hypertrophic pathway, GSK-3β (estimated by its augmented phosphorylated level). In contrast, exposing the NOS1 overexpressed NRVMs to PE-treatment further increased the hypertrophic growth, ANF transcriptional activity and calcineurin activity. Together, the results of the present study suggest that NOS1 is directly involved in controlling the development of cardiomyocyte hypertrophy.

Keywords: Adenovirus; Cardiomyocyte hypertrophy; Gene silencing; NFAT; Neuronal NO synthase.

Fig. 1

Selective neuronal nitric oxide synthase inhibition blocks cardiomyocyte hypertrophy in vitro.A, Representative immunoblot showing NOS1 expression in response to PE stimulation. Values are expressed as mean ± SEM of triplicates from three independent experiments. B, Representative immunofluorescence of alpha-actinin (green) and nuclear staining (blue) and corresponding quantification of the cardiomyocyte cell surface area for each treatment condition. Scale bar = 50 μm. C, Tritiated leucine incorporation as a function of LVNIO (10⁻⁶ Mol/L) and/or phenylephrine treatments. Results are expressed as percentage activation of control. *P < 0.05 versus non treated cells, ^#P < 0.01 versus PE. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Modulation of NOS1 expression by specific siRNA or adenovirus is efficient into neonatal rat cardiomyocytes. Representative immunoblots and quantification for NOS1 and GAPDH of NRVMs treated with si-NOS1, Ad.NOS1 and their respective controls. Values are expressed as mean ± SEM from six independent experiments. *P < 0.05 versus non treated cells.

Fig. 3

NOS1 is involved in PE-induced NRVMs hypertrophy. A, Representative immunofluorescence images showing α-actinin (green) and DAPI (blue) -stained neonatal rat cardiomyocytes and bar graph depicting average cell surface area for each treatment condition. Scale bar, 50 μm. B, Tritiated leucine incorporation as a function of PE treatment and regulation of NOS1 protein expression using si-NOS1 or Ad.NOS1. C, NRVMS cotransfected with ANF-Luc and si-NOS1 or Ad.NOS1 were treated or not with PE for 48 h and were assayed for luciferase activity. Results are expressed as percentage activation of control. Values are expressed as mean ± SEM of triplicates from three independent experiments. *P < 0.05 versus non treated cells, ^#P < 0.05 versus PE. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

NOS1 effect on cardiomyocyte hypertrophy is mediated by the NFAT signaling pathway. A, Representative immunoblots for calcineurin and its corresponding quantification normalized to GAPDH. B, NRVMS cotransfected with NFAT-Luc and si-NOS1 or Ad.NOS1 were treated or not with PE for 48 h and were assayed for luciferase activity. Results are expressed as percentage activation of control. C, Representative phospho-GSK-3β and total GSK-3β immunoblots and corresponding level of activation determined by the ratio phospho to total GSK-3β normalized to GAPDH. Values are expressed as mean ± SEM of triplicates from three independent experiments. *P < 0.05 versus the non-treated cells, ^#P < 0.05 versus PE.