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Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiomyocyte hypertrophy.

Mohamed IA, Gadeau AP, Fliegel L, Lopaschuk G, Mlih M, Abdulrahman N, Fillmore N, Mraiche F - PLoS ONE (2015)

Bottom Line: Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4.NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1.Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Qatar University, Doha, Qatar.

ABSTRACT
Enhanced expression and activity of the Na+/H+ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.

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OPN siRNA regresses expression of the GATA-4 hypertrophic transcription factor.Upper panel, representative western blot of relative amounts of phosphorylated and total GATA4 expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 in the presence and absence of siRNA OPN. Lower panel: quantification of experiments measuring the ratio of phosphorylated to total GATA4 protein. Results are expressed as % of control (GFP) ± %SEM (n = 4–7; representative of 3–5 experiments). *p < 0.05 vs. control, # vs. NHE1.
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pone.0123318.g003: OPN siRNA regresses expression of the GATA-4 hypertrophic transcription factor.Upper panel, representative western blot of relative amounts of phosphorylated and total GATA4 expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 in the presence and absence of siRNA OPN. Lower panel: quantification of experiments measuring the ratio of phosphorylated to total GATA4 protein. Results are expressed as % of control (GFP) ± %SEM (n = 4–7; representative of 3–5 experiments). *p < 0.05 vs. control, # vs. NHE1.

Mentions: The NHE1-mediated hypertrophic effect has previously been demonstrated to be driven at least in part by the CaN/NFAT pathway [25,26]. We tested whether OPN contributes to this pathway by examining the level of GATA4 phosphorylation, a key activator of the CaN/NFAT pathway that leads to the expression of hypertrophic genes. Active NHE1 enhanced phosphorylation of GATA4 (144.0±14.01% of control; P<0.05) (Fig 3). GATA4 phosphorylation was significantly reduced following inhibition of OPN by transfection of cardiomyocytes with siRNA compared to cardiomyocytes expressing active NHE1 (93.7±6.31% vs. 144.0±14.01% NHE1; P< 0.05). Our findings revealed for the first time that NHE1 induced OPN expression regulated hypertrophic gene transcription.


Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiomyocyte hypertrophy.

Mohamed IA, Gadeau AP, Fliegel L, Lopaschuk G, Mlih M, Abdulrahman N, Fillmore N, Mraiche F - PLoS ONE (2015)

OPN siRNA regresses expression of the GATA-4 hypertrophic transcription factor.Upper panel, representative western blot of relative amounts of phosphorylated and total GATA4 expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 in the presence and absence of siRNA OPN. Lower panel: quantification of experiments measuring the ratio of phosphorylated to total GATA4 protein. Results are expressed as % of control (GFP) ± %SEM (n = 4–7; representative of 3–5 experiments). *p < 0.05 vs. control, # vs. NHE1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401699&req=5

pone.0123318.g003: OPN siRNA regresses expression of the GATA-4 hypertrophic transcription factor.Upper panel, representative western blot of relative amounts of phosphorylated and total GATA4 expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 in the presence and absence of siRNA OPN. Lower panel: quantification of experiments measuring the ratio of phosphorylated to total GATA4 protein. Results are expressed as % of control (GFP) ± %SEM (n = 4–7; representative of 3–5 experiments). *p < 0.05 vs. control, # vs. NHE1.
Mentions: The NHE1-mediated hypertrophic effect has previously been demonstrated to be driven at least in part by the CaN/NFAT pathway [25,26]. We tested whether OPN contributes to this pathway by examining the level of GATA4 phosphorylation, a key activator of the CaN/NFAT pathway that leads to the expression of hypertrophic genes. Active NHE1 enhanced phosphorylation of GATA4 (144.0±14.01% of control; P<0.05) (Fig 3). GATA4 phosphorylation was significantly reduced following inhibition of OPN by transfection of cardiomyocytes with siRNA compared to cardiomyocytes expressing active NHE1 (93.7±6.31% vs. 144.0±14.01% NHE1; P< 0.05). Our findings revealed for the first time that NHE1 induced OPN expression regulated hypertrophic gene transcription.

Bottom Line: Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4.NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1.Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Qatar University, Doha, Qatar.

ABSTRACT
Enhanced expression and activity of the Na+/H+ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.

Show MeSH
Related in: MedlinePlus