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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.A: upper panel, representative fluorescence microscopy images of NRVMs infected with GFP (control) or active NHE1 adenovirus 24 hours post infection; lower panel, cell surface area of at least 50–70 infected NRVMs from 4–6 individual dishes were measured to represent 3–4 experiments, results expressed as % of control (GFP) ± %SEM. B: Upper panel, representative fluorescence microscopy images of H9c2 cardiomyocytes infected with adenoviruses containing GFP (control) or active NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, cell surface area of at least 50–70 H9c2 cardiomyocytes from 4–6 individual dishes were measured to represent 3–4 experiments. C: Protein content of H9c2 cardiomyocytes expressed as μg/10 x 106 cell. D: Quantification of ANP mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.
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pone.0123318.g002: OPN contributes to NHE1-induced cardiomyocyte-hypertrophy.A: upper panel, representative fluorescence microscopy images of NRVMs infected with GFP (control) or active NHE1 adenovirus 24 hours post infection; lower panel, cell surface area of at least 50–70 infected NRVMs from 4–6 individual dishes were measured to represent 3–4 experiments, results expressed as % of control (GFP) ± %SEM. B: Upper panel, representative fluorescence microscopy images of H9c2 cardiomyocytes infected with adenoviruses containing GFP (control) or active NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, cell surface area of at least 50–70 H9c2 cardiomyocytes from 4–6 individual dishes were measured to represent 3–4 experiments. C: Protein content of H9c2 cardiomyocytes expressed as μg/10 x 106 cell. D: Quantification of ANP mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.

Mentions: Although OPN has been suggested to mediate CH [17–19], whether OPN contributes to cardiomyocyte hypertrophy induced by elevated expression and activity of NHE1 has not been shown. In vitro, cardiomyocytes infected with the OPN adenovirus alone (expressing a three fold increase in OPN mRNA vs. control) did not cause a significant increase in cell surface area (153.2±26.65% of control), protein content (156.5±19.86% of control) or ANP mRNA (141.5±86.65% of control). Transfection of cardiomyocytes with siRNA-OPN alone (in the absence of the NHE1 adenovirus) was also unable to reverse any parameters of cardiomyocyte hypertrophy compared to control (cell surface area (51.9±1170.77% of control), protein content (96.8±17.81% of control) or ANP mRNA (66.3±0.59% of control)). However, cardiomyocytes expressing the active form of NHE1 adenovirus induced cardiomyocyte hypertrophy as indicated by the significant increase in cell area in both the H9c2 cardiomyocytes and NRVMs (Fig 2A and 2B). Total protein content (Fig 2D) and ANP mRNA expression (Fig 2E) were also significantly increased (136.8±11% and 247.7±30.81% of control; P<0.05 respectively) in H9c2 cardiomyocytes expressing the active form of the NHE1 adenovirus. The downregulation of OPN by transfection of siRNA directed against OPN in cardiomyocytes expressing active NHE1 fully reversed the NHE1 hypertrophic effect as indicated by the significant reduction in cell surface area (68.5±0.24% vs. 190.9±8.66%; P< 0.05), total protein content (87.8±12.58% vs. 136.8±11%, P< 0.05) and ANP mRNA expression (64.6±19.9% vs. 247.7±30.81% P< 0.05) (Fig 2B–2E). Transfection of H9c2 cardiomyocytes with scrambled siRNA did not alter protein content (121.3±27.48% of control). Our findings reveal for the first time that NHE1-induced OPN expression contributed to the hypertrophic response in cardiomyocytes and that partial inhibition of OPN prevented the NHE1 induced hypertrophic response.


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.A: upper panel, representative fluorescence microscopy images of NRVMs infected with GFP (control) or active NHE1 adenovirus 24 hours post infection; lower panel, cell surface area of at least 50–70 infected NRVMs from 4–6 individual dishes were measured to represent 3–4 experiments, results expressed as % of control (GFP) ± %SEM. B: Upper panel, representative fluorescence microscopy images of H9c2 cardiomyocytes infected with adenoviruses containing GFP (control) or active NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, cell surface area of at least 50–70 H9c2 cardiomyocytes from 4–6 individual dishes were measured to represent 3–4 experiments. C: Protein content of H9c2 cardiomyocytes expressed as μg/10 x 106 cell. D: Quantification of ANP mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.
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Related In: Results  -  Collection

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pone.0123318.g002: OPN contributes to NHE1-induced cardiomyocyte-hypertrophy.A: upper panel, representative fluorescence microscopy images of NRVMs infected with GFP (control) or active NHE1 adenovirus 24 hours post infection; lower panel, cell surface area of at least 50–70 infected NRVMs from 4–6 individual dishes were measured to represent 3–4 experiments, results expressed as % of control (GFP) ± %SEM. B: Upper panel, representative fluorescence microscopy images of H9c2 cardiomyocytes infected with adenoviruses containing GFP (control) or active NHE1 in the presence and absence of siRNA OPN for 24 hours; lower panel, cell surface area of at least 50–70 H9c2 cardiomyocytes from 4–6 individual dishes were measured to represent 3–4 experiments. C: Protein content of H9c2 cardiomyocytes expressed as μg/10 x 106 cell. D: Quantification of ANP mRNA expression in H9c2 cardiomyocytes normalized to β-actin (n = 6–7; representative of 4 experiments). Results are expressed as % of control (GFP) ± %SEM. *p < 0.05 vs. control, # vs. NHE1.
Mentions: Although OPN has been suggested to mediate CH [17–19], whether OPN contributes to cardiomyocyte hypertrophy induced by elevated expression and activity of NHE1 has not been shown. In vitro, cardiomyocytes infected with the OPN adenovirus alone (expressing a three fold increase in OPN mRNA vs. control) did not cause a significant increase in cell surface area (153.2±26.65% of control), protein content (156.5±19.86% of control) or ANP mRNA (141.5±86.65% of control). Transfection of cardiomyocytes with siRNA-OPN alone (in the absence of the NHE1 adenovirus) was also unable to reverse any parameters of cardiomyocyte hypertrophy compared to control (cell surface area (51.9±1170.77% of control), protein content (96.8±17.81% of control) or ANP mRNA (66.3±0.59% of control)). However, cardiomyocytes expressing the active form of NHE1 adenovirus induced cardiomyocyte hypertrophy as indicated by the significant increase in cell area in both the H9c2 cardiomyocytes and NRVMs (Fig 2A and 2B). Total protein content (Fig 2D) and ANP mRNA expression (Fig 2E) were also significantly increased (136.8±11% and 247.7±30.81% of control; P<0.05 respectively) in H9c2 cardiomyocytes expressing the active form of the NHE1 adenovirus. The downregulation of OPN by transfection of siRNA directed against OPN in cardiomyocytes expressing active NHE1 fully reversed the NHE1 hypertrophic effect as indicated by the significant reduction in cell surface area (68.5±0.24% vs. 190.9±8.66%; P< 0.05), total protein content (87.8±12.58% vs. 136.8±11%, P< 0.05) and ANP mRNA expression (64.6±19.9% vs. 247.7±30.81% P< 0.05) (Fig 2B–2E). Transfection of H9c2 cardiomyocytes with scrambled siRNA did not alter protein content (121.3±27.48% of control). Our findings reveal for the first time that NHE1-induced OPN expression contributed to the hypertrophic response in cardiomyocytes and that partial inhibition of OPN prevented the NHE1 induced hypertrophic response.

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