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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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Related in: MedlinePlus

Upregulation of NHE1 in cardiomyocytes enhances OPN expression.A: Upper panel, representative western blot of OPN protein expression in cell lysates of NRVMs infected with GFP (control) or active NHE1 adenovirus for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and GAPDH (38 kDa). Lower level, quantification of relative levels of total OPN protein expression (n = 4; representative of 2–3 preparations). B: Upper panel, representative DNA gel of OPN mRNA expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 adenovirus for 24 hours using an MOI of 20 and 30, respectively. cDNA amplification was against OPN (206 bp) and β-actin (174 bp). Lower level, quantification of OPN mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control. C: Upper panel, representative western blot of OPN protein expression of H9c2 cardiomyocytes infected with NHE1 adenovirus (30 MOI) in the presence and absence of 100nM siRNA OPN for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 6–9; representative of 2–3 experiments). Results are expressed as % of NHE1 ± %SEM. *p < 0.05 vs. NHE1 + siRNA.
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pone.0123318.g001: Upregulation of NHE1 in cardiomyocytes enhances OPN expression.A: Upper panel, representative western blot of OPN protein expression in cell lysates of NRVMs infected with GFP (control) or active NHE1 adenovirus for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and GAPDH (38 kDa). Lower level, quantification of relative levels of total OPN protein expression (n = 4; representative of 2–3 preparations). B: Upper panel, representative DNA gel of OPN mRNA expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 adenovirus for 24 hours using an MOI of 20 and 30, respectively. cDNA amplification was against OPN (206 bp) and β-actin (174 bp). Lower level, quantification of OPN mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control. C: Upper panel, representative western blot of OPN protein expression of H9c2 cardiomyocytes infected with NHE1 adenovirus (30 MOI) in the presence and absence of 100nM siRNA OPN for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 6–9; representative of 2–3 experiments). Results are expressed as % of NHE1 ± %SEM. *p < 0.05 vs. NHE1 + siRNA.

Mentions: Previous reports have demonstrated a simultaneous upregulation of NHE1 and OPN in models of CH [5,6,15,16]. In order to determine whether NHE1 activation induces OPN expression during cardiomyocyte hypertrophy, H9c2 cardiomyocytes and NRVMs were infected with adenoviral vectors coding a constitutively active NHE1 for 24 hours. An enhanced green fluorescent protein (GFP)-expressing adenovirus under the control of the same promoter of the NHE1 and OPN adenoviruses served as a control. Our results indicated that infection of H9c2 cardiomyocytes or NRVMs with the NHE1 adenovirus successfully overexpressed the HA-tagged protein (S1A Fig and S2A Fig), as well as total NHE1 protein expression (S1B Fig and S2B Fig), an effect that was not observed in cardiomyocytes infected with the GFP adenovirus alone. OPN protein and mRNA expression were then examined in cardiomyocytes infected with the active form of the NHE1 adenovirsu. OPN protein expression was examined in NRVMs 24 hours post infection. Our results revealed that expression of the OPN protein, appearing as a doublet at 66 kDa [17,40], was significantly elevated in cardiomyocytes expressing the active form of the NHE1 adenovirus (342.7%±69.22% vs. 100.0±33.93% control; P<0.05 (Fig 1A). OPN mRNA expression was significantly increased in cardiomyocytes expressing active NHE1 in H9c2 cardiomyocytes (296.3%±53.60% vs. 100.0% control; P<0.05) (Fig 1B). To identify the role of OPN in the NHE1 mediated hypertrophic effect we induced NHE1 activity in cardiomyocytes and blocked the OPN using siRNA (S3B Fig). The remaining portion of our study in which OPN was blocked using siRNA was carried out in H9c2 cardiomyocytes. The H9c2 cell model has been suggested to be a more suitable host for siRNA transfection as it represents a form of immortal mammalian cell line as opposed to NRVMs, which may require electroporation to allow for the uptake of siRNA [41]. Inhibition of OPN with siRNA directed against OPN reduced the NHE1-induced upregulation of OPN protein expression by more than 50% in H9c2 cardiomyocytes at the 24 hour time point (49.5±9.18% vs. 100.0% NHE1; P< 0.05) (Fig 1C and S3A Fig). Our results demonstrated for the first time that the upregulation of active NHE1 in cardiomyocytes induced the expression of OPN in both NRVMs and H9c2 cardiomyocytes.


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)

Upregulation of NHE1 in cardiomyocytes enhances OPN expression.A: Upper panel, representative western blot of OPN protein expression in cell lysates of NRVMs infected with GFP (control) or active NHE1 adenovirus for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and GAPDH (38 kDa). Lower level, quantification of relative levels of total OPN protein expression (n = 4; representative of 2–3 preparations). B: Upper panel, representative DNA gel of OPN mRNA expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 adenovirus for 24 hours using an MOI of 20 and 30, respectively. cDNA amplification was against OPN (206 bp) and β-actin (174 bp). Lower level, quantification of OPN mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control. C: Upper panel, representative western blot of OPN protein expression of H9c2 cardiomyocytes infected with NHE1 adenovirus (30 MOI) in the presence and absence of 100nM siRNA OPN for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 6–9; representative of 2–3 experiments). Results are expressed as % of NHE1 ± %SEM. *p < 0.05 vs. NHE1 + siRNA.
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pone.0123318.g001: Upregulation of NHE1 in cardiomyocytes enhances OPN expression.A: Upper panel, representative western blot of OPN protein expression in cell lysates of NRVMs infected with GFP (control) or active NHE1 adenovirus for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and GAPDH (38 kDa). Lower level, quantification of relative levels of total OPN protein expression (n = 4; representative of 2–3 preparations). B: Upper panel, representative DNA gel of OPN mRNA expression in H9c2 cardiomyocytes infected with GFP (control) or active NHE1 adenovirus for 24 hours using an MOI of 20 and 30, respectively. cDNA amplification was against OPN (206 bp) and β-actin (174 bp). Lower level, quantification of OPN mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control. C: Upper panel, representative western blot of OPN protein expression of H9c2 cardiomyocytes infected with NHE1 adenovirus (30 MOI) in the presence and absence of 100nM siRNA OPN for 24 hours. Immunoblotting was against OPN (doublet at 66 kDa) and α-tubulin (50 kDa); lower panel, quantification of relative levels of OPN protein expression (n = 6–9; representative of 2–3 experiments). Results are expressed as % of NHE1 ± %SEM. *p < 0.05 vs. NHE1 + siRNA.
Mentions: Previous reports have demonstrated a simultaneous upregulation of NHE1 and OPN in models of CH [5,6,15,16]. In order to determine whether NHE1 activation induces OPN expression during cardiomyocyte hypertrophy, H9c2 cardiomyocytes and NRVMs were infected with adenoviral vectors coding a constitutively active NHE1 for 24 hours. An enhanced green fluorescent protein (GFP)-expressing adenovirus under the control of the same promoter of the NHE1 and OPN adenoviruses served as a control. Our results indicated that infection of H9c2 cardiomyocytes or NRVMs with the NHE1 adenovirus successfully overexpressed the HA-tagged protein (S1A Fig and S2A Fig), as well as total NHE1 protein expression (S1B Fig and S2B Fig), an effect that was not observed in cardiomyocytes infected with the GFP adenovirus alone. OPN protein and mRNA expression were then examined in cardiomyocytes infected with the active form of the NHE1 adenovirsu. OPN protein expression was examined in NRVMs 24 hours post infection. Our results revealed that expression of the OPN protein, appearing as a doublet at 66 kDa [17,40], was significantly elevated in cardiomyocytes expressing the active form of the NHE1 adenovirus (342.7%±69.22% vs. 100.0±33.93% control; P<0.05 (Fig 1A). OPN mRNA expression was significantly increased in cardiomyocytes expressing active NHE1 in H9c2 cardiomyocytes (296.3%±53.60% vs. 100.0% control; P<0.05) (Fig 1B). To identify the role of OPN in the NHE1 mediated hypertrophic effect we induced NHE1 activity in cardiomyocytes and blocked the OPN using siRNA (S3B Fig). The remaining portion of our study in which OPN was blocked using siRNA was carried out in H9c2 cardiomyocytes. The H9c2 cell model has been suggested to be a more suitable host for siRNA transfection as it represents a form of immortal mammalian cell line as opposed to NRVMs, which may require electroporation to allow for the uptake of siRNA [41]. Inhibition of OPN with siRNA directed against OPN reduced the NHE1-induced upregulation of OPN protein expression by more than 50% in H9c2 cardiomyocytes at the 24 hour time point (49.5±9.18% vs. 100.0% NHE1; P< 0.05) (Fig 1C and S3A Fig). Our results demonstrated for the first time that the upregulation of active NHE1 in cardiomyocytes induced the expression of OPN in both NRVMs and H9c2 cardiomyocytes.

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