Stimulation of Na⁺/K⁺-ATPase with an Antibody against Its 4thExtracellular Region Attenuates Angiotensin II-Induced H9c2 Cardiomyocyte Hypertrophy via an AMPK/SIRT3/PPARγSignaling Pathway

Figures

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  Figure 1. Effects of DR-Ab on Ang II-induced H9c2 cardiomyocyte hypertrophy. DR-Ab (2 μM) was given 30 min before treatment with Ang II (100 nM) for 48 h. (a, b) Representative immunofluorescence staining (a) and group data (b) showing that DR-Ab reversed enlarged cell size caused by Ang II. Red: α-actinin. Blue: DAPI. Scale bar, 25 μm. n = 6. (c) qRT-PCR analysis showing the mRNA levels of ANP, BNP, and β-MHC. n = 4. (d–g) DR-Ab reversed Ang II-induced loss of plasma membrane NKA α1 (e, f), increase of endosome NKA α1 (g), and downregulation of NKA activity (d). n = 4~6. Scale bar, 30 μm. Blue: DAPI. Green: NKA α1 staining. ∗p < 0.05 versus control group, †p < 0.05 versus Ang II alone group.

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  Figure 2. Effects of DR-Ab on Ang II-induced intracellular ROS generation in H9c2 cells. DR-Ab (2 μM) was given 30 min before treatment with Ang II (100 nM) for 48 h. (a–c) Representative immunofluorescence image (a) and group data (b, c) showing that DR-Ab decreased ROS generation caused by Ang II. Red: DHE staining (a, upper). Scale bar, 100 μm. Green: DCFH-DA staining (a, lower). Scale bar, 50 μm. n = 6. (d) Effect of DR-Ab on the protein level of two subunits of NADPH oxidase: p22phox and p47phox. n = 4‐6. ∗p < 0.05 versus control group, †p < 0.05 versus Ang II alone group.

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  Figure 3. Effect of DR-Ab on Ang II-induced mitochondrial ROS (mit-ROS) generation and energy metabolic dysfunction. (a, b) Representative image (a) and group data (b) showing that DR-Ab decreased Ang II-induced mit-ROS generation. Red: Mit-ROS. Scale bar, 50 μm. n = 4‐6. (c) Representative JC-1 staining showing that DR-Ab reversed mitochondrial membrane potential loss caused by Ang II. Red: aggregate. Green: monomer. Scale bar, 50 μm. (d) qRT-PCR analysis showing that DR-Ab increased the mRNA expression of mitochondrial encoded genes (ND-1, Cyt-b, and mt-co1) in Ang II-treated cells. n = 4. (e) qRT-PCR analysis showing the effect of DR-Ab on the mRNA expression of fatty acid oxidation related genes (CPT-1β, CPT-2, LCAD, and MCAD). n = 4. ∗p < 0.05 versus control group, †p < 0.05 versus Ang II alone group.

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  Figure 4. Effect of DR-Ab on myocyte hypertrophy and intracellular ROS generation in Ang II-treated H9c2 in the presence and absence of compound C (40 μM, a selective AMPK inhibitor), 3-TYP (50 μM, a selective Sirt3 inhibitor), or GW9662 (10 μM, a PPARγ antagonist). Cells were treated with these inhibitors for 30 min before DR-Ab (2 μM, 30 min) and Ang II (100 nM, 48 h). (a, b) Representative immunofluorescence staining (a) and group data (b) showing that blockade of AMPK, Sirt3, or PPARγ abolished the effect of DR-Ab on cell size. Red: α-actinin. Blue: DAPI. Scale bar, 25 μm. n = 4‐6. (c–f) Representative image (c, e) and group data (d, f) showing that blockade of AMPK, Sirt3, or PPARγ promoted the intracellular ROS which were decreased by DR-Ab in Ang II-induced cells. (c) Red: DHE relative fluorescence density. Scale bar, 100 μm. (e) Green: DCFH-DA staining. Scale bar, 50 μm. n = 4‐6. ∗p < 0.05 versus control, †p < 0.05 versus Ang II alone group, ‡p < 0.05 versus Ang II+DR-Ab group.

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  Figure 5. Effects of DR-Ab on mit-ROS production and energy metabolic dysfunction in Ang II-treated cells in the presence and absence of inhibitors of AMPK, Sirt3, or PPARγ. (a, b) Representative image (a) and statistic data (b) showing that blockade of AMPK, Sirt3, or PPARγ with their inhibitors abolished the protective effect of DR-Ab on mit-ROS production. Red: mit-ROS. Scale bar, 50 μm. n = 4‐6. (c) JC-1 staining showing that blockade of AMPK, Sirt3, or PPARγ reversed the effect of DR-Ab on mitochondrial membrane potential. Red: aggregate. Green: monomer. Scale bar, 50 μm. (d–h) qRT-PCR analysis showing that blockade of AMPK, Sirt3, or PPARγ abolished the effects of DR-Ab on the mRNA expression of ND-1, Cyt-b, CPT-2, LCAD, and MCAD. n = 4‐6. ∗p < 0.05 versus control, †p < 0.05 versus Ang II alone group, ‡p < 0.05 versus Ang II+DR-Ab group.

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  Figure 6. Western blotting analysis showing that DR-Ab stimulated AMPK/Sirt-3/PPARγ signaling pathway. (a) Time-course study showing the effect of DR-Ab on AMPK phosphorylation. (b–d) DR-Ab reversed the effect of Ang II on p-AMPK (b), PPARγ (c), & Sirt-3 (d). n = 4‐6. (e) 3-TYP eliminated DR-Ab effect on the PPARγ level in Ang II-treated cells. n = 4. (f, g) Compound C abolished the effect of DR-Ab on the protein expression of PPARγ (f) and Sirt-3 (g) in Ang II-treated cells. n = 4‐6. ∗p < 0.05 versus control group, †p < 0.05 versus Ang II alone group, ‡p < 0.05 versus Ang II+ DR-Ab.

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  Figure 7. Schematic illustration showing the mechanisms for the protective effects of DR-Ab. DR-Ab protects against Ang II-induced cell injury by stabilization of membrane NKA and stimulation of its activity. This helps to maintain the normal intracellular ion homeostasis, thus reserves the cell size. DR-Ab inhibits NADPH oxidase activity by downregulation of p22phox and p47phox expression. Meanwhile, DR-Ab inhibits mitochondrial ROS generation and preserves mitochondrial function through stimulation of the AMPK/Sirt-3/PPARγ signaling pathway.