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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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Downregulating OPN reduces NHE1-induced cardiomyocyte-hypertrophy and NHE1 activity.Cardiomyocytes plated on coverslips were incubated with BCECF-AM and induced with an acid load using 50 mM NH4Cl. The rate of recovery following the acid induction was measured and used as an indicator of NHE1 activity. Upper panel, representative traces of NHE1 activity assay in H9c2 cardiomyocytes infected with GFP (control) or NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, quantification of NHE1 activity (10–14 coverslips, from 3–4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.
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pone.0123318.g004: Downregulating OPN reduces NHE1-induced cardiomyocyte-hypertrophy and NHE1 activity.Cardiomyocytes plated on coverslips were incubated with BCECF-AM and induced with an acid load using 50 mM NH4Cl. The rate of recovery following the acid induction was measured and used as an indicator of NHE1 activity. Upper panel, representative traces of NHE1 activity assay in H9c2 cardiomyocytes infected with GFP (control) or NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, quantification of NHE1 activity (10–14 coverslips, from 3–4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.

Mentions: To ascertain the involvement of OPN on enhancing/maintaining NHE1 activity, NHE1 activity was measured in cardiomyocytes expressing active NHE1 in the presence and absence of OPN siRNA (Fig 4). NHE1 activity in cardiomyocytes infected with active NHE1 was significantly increased compared to control (586.5±103.54% of control; P<0.05). NHE1 activity was reduced by more than 75% by downregulating OPN expression in cardiomyocytes (235.0±92.14% vs. 586.5±103.54% NHE1; P< 0.05). These data demonstrated that the expression of OPN in cardiomyocytes contributed to NHE1 activity suggesting that OPN played a role in enhancing/maintaining NHE1 activity.


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)

Downregulating OPN reduces NHE1-induced cardiomyocyte-hypertrophy and NHE1 activity.Cardiomyocytes plated on coverslips were incubated with BCECF-AM and induced with an acid load using 50 mM NH4Cl. The rate of recovery following the acid induction was measured and used as an indicator of NHE1 activity. Upper panel, representative traces of NHE1 activity assay in H9c2 cardiomyocytes infected with GFP (control) or NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, quantification of NHE1 activity (10–14 coverslips, from 3–4 experiments). Results are expressed as % of control (GFP) ± %SEM. *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.g004: Downregulating OPN reduces NHE1-induced cardiomyocyte-hypertrophy and NHE1 activity.Cardiomyocytes plated on coverslips were incubated with BCECF-AM and induced with an acid load using 50 mM NH4Cl. The rate of recovery following the acid induction was measured and used as an indicator of NHE1 activity. Upper panel, representative traces of NHE1 activity assay in H9c2 cardiomyocytes infected with GFP (control) or NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, quantification of NHE1 activity (10–14 coverslips, from 3–4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.
Mentions: To ascertain the involvement of OPN on enhancing/maintaining NHE1 activity, NHE1 activity was measured in cardiomyocytes expressing active NHE1 in the presence and absence of OPN siRNA (Fig 4). NHE1 activity in cardiomyocytes infected with active NHE1 was significantly increased compared to control (586.5±103.54% of control; P<0.05). NHE1 activity was reduced by more than 75% by downregulating OPN expression in cardiomyocytes (235.0±92.14% vs. 586.5±103.54% NHE1; P< 0.05). These data demonstrated that the expression of OPN in cardiomyocytes contributed to NHE1 activity suggesting that OPN played a role in enhancing/maintaining NHE1 activity.

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