Betulin Protects Against Cardiac Hypertrophy by Improving AMPK /Nrf2‐Dependent Mitochondrial Function

Figures

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  FIGURE 1. Betulin attenuates Ang II‐induced hypertrophy and fibrosis in NRCMs. (A) Chemical structure of betulin. (B) Cell viability of NRCMs after betulin stimulation at various concentrations (0.1, 0.5, 2.5, 12.5, 62.5 and 312.5 μM). NRCMs were treated with betulin (10 and 20 μM) followed by Ang II stimulation for 24 h. (C–F) mRNA levels of Anp, Bnp, Col1a1 and Tgfb. (G) Representative images of TRITC‐phalloidin staining (left) and quantification of cell area (right). Scale bar, 100 μm. Data are presented as mean ± SEM (n = 3). p Values indicated. NS, no significance.

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  FIGURE 2. Betulin alleviates Ang II‐induced cardiac hypertrophy. (A) Illustration of experimental protocols. Mice were infused with Ang II at a rate of 0.11 μL/h for 4 weeks, and betulin (15 or 30 mg/kg) was administered during the last 2 weeks. After 4 weeks, the cardiac function of the mice was evaluated, and blood samples and heart tissues were harvested. (B) Systolic blood pressure of mice was measured during the study. *p < 0.001 versus Sham; NS, no significance. (C–E) Serum levels of Ang II, ANP and CK‐MB were detected using ELISA kits. (F–G) mRNA levels of Anp and Bnp in heart tissue. (H) Representative left ventricular M‐mode echocardiographic images. (I–M) Values of diastole LVIDd, IVSD, EF, FS and HR. (N) Representative images of the gross heart. (O–P) Values of heart weight to body weight ratios (HW/BW), and HW/TL. (Q) Representative images of H&E staining in heart tissue samples (transverse). (R–S) Representative images and quantification of WGA staining. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 6). p Values indicated.

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  FIGURE 3. Betulin ameliorates TAC‐induced cardiac hypertrophy. (A) Schematic representation of the experimental design for the TAC‐induced cardiac remodeling model. (B, C) Serum concentrations of ANP and CK‐MB. (D, E) mRNA levels of Anp and Myh7 were detected by real‐time quantitative polymerase chain reaction (RT–qPCR) analysis in heart tissues. (F) Representative M‐mode echocardiographic images. (G–K) Values of diastole LVIDd, IVSD, EF, FS and HR. (L) Representative images of the whole heart. (M–N) Ratios of HW/BW and HW/TL. (O–Q) Representative images of H&E and WGA staining in heart samples, and quantification of WGA staining. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 6). p Values indicated.

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  FIGURE 4. Betulin exerts cardioprotective effects by activating the AMPK/Nrf2 pathway. (A, B) Two weeks after betulin (30 mg/kg) administration, the heart was collected for in vitro RNA sequencing analysis. Volcano maps revealed the DEGs between the Ang II group versus the sham group (A) and the Ang II + betulin group versus the Ang II group (B). (C, D) KEGG enrichment of DEGs in Ang II group versus Sham group (C) and Ang II group versus Ang II + betulin group (D). (E, F) Activation of the AMPK and Nrf2 pathway was assessed by measuring phosphorylated AMPK, AMPK, HO‐1, NQO‐1, total Nrf2 and nuclear Nrf2 in heart tissue. (G, H) Western blotting of phosphorylated AMPK, AMPK, HO‐1, NQO‐1, total Nrf2 and nuclear Nrf2 in NRCMs. (I) Venn diagram of DEGs upregulated in the Ang II group compared to the sham group (red), and DEGs upregulated in the Ang II group compared to the Ang II + betulin group (blue). (J) Transcription factor prediction was performed. Scale bar, 100 μm. Data are revealed as mean ± SEM (n = 3). p Values indicated. Nuc, nuclear.

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  FIGURE 5. Betulin mediates Ang II‐challenged oxidative stress and mitochondrial injury. (A–D) Representative images of DHE staining (red) and quantification in heart tissues and NRCMs. (E) Representative transmission electron micrographs of NRCMs, and mitochondrial scores were analyzed. (F) Representative images of JC‐1 staining in NRCMs. (G) ATP contents in NRCMs. (H, I) Oxygen consumption by mitochondrial complex I and complex II in NRCMs. Scale bar, 50 μm. Data are presented as mean ± SEM (B, n = 4; D, n = 3). p Values indicated.

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  FIGURE 6. Inhibition of the AMPK pathway suppresses the cardioprotective effects of betulin in Ang II‐challenged NRCMs. (A) NRCMs were treated with betulin (20 μM) and compound C (2 μM), followed by Ang II stimulation for 24 h. Representative images of TRITC‐phalloidin staining (left) and quantification of cell area (right). (B) Expression levels of total Nrf2 and nuclear Nrf2 were detected by Western blotting, with quantitation on the right. (C, D) Representative images of DHE staining (red) and quantification. (E, F) Representative images of JC‐1 staining and the red‐to‐green fluorescence ratio. (G, H) Representative images of TRITC‐phalloidin staining and quantification of cell area in NRCMs from the indicated groups. (I, J) Representative images of DHE staining (red) and quantification of NRCMs in the indicated groups. Scale bar, A, 100 μm; C and E, 50 μm. Data are presented as mean ± SEM (n = 3). p Values indicated. Nuc, nuclear.

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  FIGURE 7. AMPKα2 knockout diminishes the cardioprotective effects of betulin in Ang II‐challenged mice. (A) Schematic representation of the experimental protocols. Wild‐type (WT) mice and AMPKα2 knockout (AMPKα2−/−) mice were infused with Ang II via an osmotic pump for 4 weeks. Betulin (30 mg/kg) was administered during the last 2 weeks. After 4 weeks, cardiac function was assessed using echocardiography, and the mice were euthanized for sample collection. (B–D) Serum levels of Ang II, ANP and CK‐MB. (E, F) Real‐time quantitative PCR analysis of mRNA levels of Anp and Bnp in cardiac tissues. (G) Representative M‐mode echocardiographic images. (H–K) Values of EF, FS, LVIDd, and IVSD. (L) Representative images of the whole heart. (M) Ratio of HW/TL. (N–O) Representative images of Masson staining, with quantitative analysis. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 6). p Values indicated.