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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 contributes to NHE1-induced cardiomyocyte-hypertrophy through RSK.A. Upper panel: Representative western blot of relative amounts of phosphorylated and total expression of RSK in NRVMs infected with GFP (control) or active NHE1. Immunoblotting was against phosphorylated and total RSK (80–90 kDa) and normalized to GAPDH (38 kDa); lower panel, quantification of experiments measuring the ratio of phosphorylated to total protein for RSK. Results are expressed as % of control (GFP) ± %SEM (n = 4; representative of 2 preparations). B. H9c2 cardiomyoblasts were treated with PE and/or BI-D1870 (10 μM) for 24 h. Cells were lysed and equal amounts of protein were analyzed by SDS-PAGE/immunoblot Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 4–5; representative of 5 experiments). Results are expressed as % of control ± %SEM. *p < 0.05 vs. control.
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pone.0123318.g005: OPN contributes to NHE1-induced cardiomyocyte-hypertrophy through RSK.A. Upper panel: Representative western blot of relative amounts of phosphorylated and total expression of RSK in NRVMs infected with GFP (control) or active NHE1. Immunoblotting was against phosphorylated and total RSK (80–90 kDa) and normalized to GAPDH (38 kDa); lower panel, quantification of experiments measuring the ratio of phosphorylated to total protein for RSK. Results are expressed as % of control (GFP) ± %SEM (n = 4; representative of 2 preparations). B. H9c2 cardiomyoblasts were treated with PE and/or BI-D1870 (10 μM) for 24 h. Cells were lysed and equal amounts of protein were analyzed by SDS-PAGE/immunoblot Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 4–5; representative of 5 experiments). Results are expressed as % of control ± %SEM. *p < 0.05 vs. control.

Mentions: Several kinases including ERK 1/2, RSK and Akt have been shown to be induced during conditions of CH [42]. Whether NHE1 induced OPN expression in cardiomyocytes is mediated in part by the MAPK signaling pathway has not been demonstrated. To further understand how active NHE1 increases OPN expression in cardiomyocytes, we measured the expression of the phosphorylated and total proteins of ERK 1/2 (S4 Fig), Akt (S4 Fig) and RSK (Fig 5A) following 24 hours of infection with the active NHE1 adenovirus. The ratio of phosphorylated to total ERK 1/2 and Akt were not significantly different in cardiomyocytes expressing active NHE1 (S4 Fig). Similarly, cardiomyocytes infected with the active form of the NHE1 adenovirus demonstrated a trend towards increase in the ratio of phosphorylated to total RSK (138.2±43.02% of control), but not a significant increase. Further studies were carried out to delineate the effects of RSK in the NHE1-induced OPN hypertrophic response following a time dependent stimulation with PE, a known NHE1 stimulator [43], in the presence of BI-D1870 (Fig 5B). Our findings revealed that PE induced the expression of OPN following 30 minutes of PE stimulation, an effect that was significantly reduced in the presence of BI-D1870 (53.7±10.45% vs. of control; P< 0.05). These results suggested that the NHE1 induced OPN expression may in part be dependent on RSK.


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 contributes to NHE1-induced cardiomyocyte-hypertrophy through RSK.A. Upper panel: Representative western blot of relative amounts of phosphorylated and total expression of RSK in NRVMs infected with GFP (control) or active NHE1. Immunoblotting was against phosphorylated and total RSK (80–90 kDa) and normalized to GAPDH (38 kDa); lower panel, quantification of experiments measuring the ratio of phosphorylated to total protein for RSK. Results are expressed as % of control (GFP) ± %SEM (n = 4; representative of 2 preparations). B. H9c2 cardiomyoblasts were treated with PE and/or BI-D1870 (10 μM) for 24 h. Cells were lysed and equal amounts of protein were analyzed by SDS-PAGE/immunoblot Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 4–5; representative of 5 experiments). Results are expressed as % of control ± %SEM. *p < 0.05 vs. control.
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pone.0123318.g005: OPN contributes to NHE1-induced cardiomyocyte-hypertrophy through RSK.A. Upper panel: Representative western blot of relative amounts of phosphorylated and total expression of RSK in NRVMs infected with GFP (control) or active NHE1. Immunoblotting was against phosphorylated and total RSK (80–90 kDa) and normalized to GAPDH (38 kDa); lower panel, quantification of experiments measuring the ratio of phosphorylated to total protein for RSK. Results are expressed as % of control (GFP) ± %SEM (n = 4; representative of 2 preparations). B. H9c2 cardiomyoblasts were treated with PE and/or BI-D1870 (10 μM) for 24 h. Cells were lysed and equal amounts of protein were analyzed by SDS-PAGE/immunoblot Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 4–5; representative of 5 experiments). Results are expressed as % of control ± %SEM. *p < 0.05 vs. control.
Mentions: Several kinases including ERK 1/2, RSK and Akt have been shown to be induced during conditions of CH [42]. Whether NHE1 induced OPN expression in cardiomyocytes is mediated in part by the MAPK signaling pathway has not been demonstrated. To further understand how active NHE1 increases OPN expression in cardiomyocytes, we measured the expression of the phosphorylated and total proteins of ERK 1/2 (S4 Fig), Akt (S4 Fig) and RSK (Fig 5A) following 24 hours of infection with the active NHE1 adenovirus. The ratio of phosphorylated to total ERK 1/2 and Akt were not significantly different in cardiomyocytes expressing active NHE1 (S4 Fig). Similarly, cardiomyocytes infected with the active form of the NHE1 adenovirus demonstrated a trend towards increase in the ratio of phosphorylated to total RSK (138.2±43.02% of control), but not a significant increase. Further studies were carried out to delineate the effects of RSK in the NHE1-induced OPN hypertrophic response following a time dependent stimulation with PE, a known NHE1 stimulator [43], in the presence of BI-D1870 (Fig 5B). Our findings revealed that PE induced the expression of OPN following 30 minutes of PE stimulation, an effect that was significantly reduced in the presence of BI-D1870 (53.7±10.45% vs. of control; P< 0.05). These results suggested that the NHE1 induced OPN expression may in part be dependent on RSK.

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