Inhibiting SHP2 improves ventricular remodeling by restoring AMPK phosphorylation and mitochondrial homeostasis

Figures

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  Fig. 1. Identification of SHP2 as a regulator of myocardial hypertrophy. A Heatmap analysis of transcriptional levels of PTPNs subfamily in heart tissues from patients with non-ischemic cardiomyopathy (NICM) and non-heart failure individuals (Ctrl). n = 6–21. B-D The mRNA levels of PTPN11 between hypertrophic cardiomyopathy (HCM) patients or heart failure (HF) patients and normal individuals (Nor). n = 4–21. E-F Ventricular remodeling was induced in mice by subcutaneous infusion of Ang II (1 µg/kg/min) or ISO (15 µg/kg/min) via osmotic minipumps for 4 weeks. The mRNA levels of Ptpn11 in the heart tissues from Ang II- or ISO-induced mice. n = 7. G-H Ventricular remodeling was induced in mice by subcutaneous infusion of Ang II (1 µg/kg/min) or ISO (15 µg/kg/min) via osmotic minipumps for 4 weeks. Representative immunoblot and quantitative analysis of SHP2 in heart tissues from Ang II- or ISO-induced mice. n = 6. I-J Representative immunoblot and quantitative analysis of SHP2 in NRCMs induced by Ang II (1 µM) or ISO (1 µM) at different times. n = 5. K Immunofluorescence staining of SHP2 (green), α-actinin (red), and DAPI (blue) in heart tissues from Ang II-induced mice. Scale bar = 200 μm.…

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  Fig. 2. Inhibition of SHP2 mitigates Ang II-induced cardiac hypertrophy and fibrosis in NRCMs. A-D The mRNA levels of Anp, Myh7, Col1a1 and Tgfb1 in NRCMs stimulated with Ang II (1µM) and treated concurrently with different concentrations of SHP099 (1 µM or 5 µM) for 24 h. n = 5. E Representative images of rhodamine phalloidin staining (left) and quantitative analysis of cell area (right) in NRCMs treated with Ang II (1µM) and co-treated with different concentrations of SHP099 (1 µM or 5 µM) for 24 h. Scale bar = 50 μm. n = 5. F-I The mRNA levels of Anp, Myh7, Col1a1 and Tgfb1 in NRCMs stimulated with Ang II (1µM) for 24 h followed by transfection with siSHP2 or SHP2OE for an additional 24 h. n = 5. J Representative images of rhodamine phalloidin staining (left) and quantitative analysis of cell area (right) in NRCMs stimulated with Ang II (1µM) for 24 h followed by transfection with siSHP2 or SHP2OE for an additional 24 h. Scale bar = 50 μm. n = 5. K-N The mRNA levels of Anp, Myh7, Col1a1 and Tgfb1. NRCMs were transfected with plasmid encoding wild type SHP2 (SHP2WT) or mutation SHP2 of cysteine to alanine at C459 (SHP2C459A), and the endogenous SHP2 in NRCMs was silenced using siSHP2,…

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  Fig. 3. Pharmacological inhibition of SHP2 prevents Ang II-induced myocardial hypertrophy and fibrosis in mice. A The schematic illustrates the experimental protocols. Mice were infused with Ang II at 0.11 µL/h via osmotic pump for 4 weeks, and treated with SHP099 (2.5 mg/kg and 5 mg/kg) in the last two weeks. After 4 weeks, cardiac function was evaluated, and blood and heart tissues were collected. B-C Systolic blood pressure and body weight over a 4-week period. n = 6. D Serum Ang II levels were detected by ELISA kit. n = 6. E Representative images of M-mode echocardiogram from each group. n = 6. F-G Statistical graphs of ejection fraction (EF%) and fractional shortening (FS%). n = 6. H-I The mRNA levels of Anp and Myh7 in the heart tissues. n = 6. J Representative images of gross heart. n = 6. K The ratios of heart weight to tibial length (HW/TL). n = 6. L Representative images and quantitative analysis of HE staining and WGA staining in heart tissues. Scale bar = 100 μm. n = 6. M Representative images and quantitative analysis of Masson staining in heart tissues. Scale bar = 100 μm. n = 6. Data are presented as mean ± SEM. B and C, Two-way ANOVA with Tukey’s multiple comparisons; D,…

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  Fig. 4. Inhibition of SHP2 by SHP099 prevents ISO-induced myocardial hypertrophy and fibrosis in mice. A The schematic illustrates the experimental protocols. Mice were infused with ISO at 0.25 µL/h via osmotic pump for 4 weeks, and treated with SHP099 (2.5 mg/kg and 5 mg/kg) in the last two weeks. After 4 weeks, cardiac function was evaluated, and blood and heart tissues were collected. B Representative images of M-mode echocardiogram from each group. n = 6. C-D Statistical graphs of EF% and FS%. n = 6. E-F The mRNA expression levels of Anp and Myh7 in the heart tissues. n = 6. G Representative gross heart images. n = 6. H The ratios of HW/TL. n = 6. I Representative images and quantitative analysis of HE staining and WGA staining in heart tissues. Scale bar = 100 μm. n = 6. J Representative images and quantitative analysis of Masson staining in heart tissues. Scale bar = 100 μm. n = 6. Data are presented as mean ± SEM. C-E and H-J, One-way ANOVA with Tukey’s multiple comparisons; F, Welch’s ANOVA with Dunnett’s T3 multiple comparisons; P values indicated

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  Fig. 5. SHP2 inhibition alleviates myocardial hypertrophy by regulating AMPK phosphorylation. A-B Volcano plot of differentially expressed genes (DEGs) in Ang II-induced mice and sham mice (A), and Ang II-induced mice with or without 5 µM SHP099-treatment (B). The Q value represents the false discovery rate (FDR)-adjusted P-value. Criteria: |log₂FC| > 1; Q<0.05 n = 3. C-D The KEGG analysis with pathways enriched with DEGs (C: Ang II vs. sham; D: Ang II + SHP099 vs. Ang II). n = 3. E Gene Set Enrichment Analysis (GSEA) plots showing the enrichment results. n = 3. F Representative immunoblot and quantitative analysis of pT172-AMPK and AMPK in heart tissues. n = 6. G Representative immunoblot and quantitative analysis of pT172-AMPK and AMPK in NRCMs. n = 5. H Immunoblotting analysis of SHP2 and pT172-AMPK protein expression changes after transfection with different doses of HA-SHP2WT. n = 5. I Immunoblotting analysis of pT172-AMPK and AMPK in NRCMs. NRCMs were transfected with SHP2WT or SHP2C459A, with endogenous SHP2 silenced using siSHP2. n = 5. J Representative images of rhodamine phalloidin staining (left) and quantification of cell area (right). NRCMs were transfected with siRNA…

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  Fig. 6. Inhibition of SHP2 improves Ang II-induced mitochondrial dysfunction. A Representative images of MitoSox staining in heart tissues. Scale bar = 50 μm. n = 6. B Representative images of transmission electron microscopy in heart tissues. n = 6. C Measurement of ATP content in heart tissues. n = 6. D Immunoblotting analysis of mitochondrial respiratory chain proteins in heart tissues. n = 6. E Flow cytometric analysis of reactive oxygen species (ROS) after DCFH-DA staining in NRCMs. n = 5. F Representative images of MitoSox staining in NRCMs. Scale bar = 200 μm. n = 5. G Measurement of ATP content in NRCMs. n = 5. H-K Measurement of mitochondrial respiration in NRCMs using the Seahorse Flux Analyzer, showing the oxygen consumption rate (OCR) under I) basal respiration, J) maximal respiration (induced by FCCP), and K) proton leak. n = 5. L Immunoblotting analysis of mitochondrial respiratory chain proteins in NRCMs. n = 5. M Immunoblotting analysis of SDHA and MTCOI in NRCMs. NRCMs were transfected with SHP2WT or SHP2C459A, with endogenous SHP2 silenced using siSHP2. n = 5. Data are presented as mean ± SEM. C, G and I-K, One-way ANOVA with Tukey’s multiple comparisons; M, Unpaired…

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  Fig. 7. SHP2 interacts with AMPK through the PTP domain. A Schematic illustration of a quantitative proteomic screen to identify proteins binding to SHP2. B Tandem mass spectrometry (MS/MS) spectrum of the specific peptide of AMPK. C-D Co-immunoprecipitation (Co-IP) of SHP2 and AMPK in heart tissues from mice treated with Ang II or ISO. n = 3. E Colocalization of SHP2 (red), AMPK (green), and DAPI (blue) in Ang II- or ISO-induced NRCMs. Scale bar = 400 μm. n = 3. F Bio-layer interferometry analysis showing interaction between SHP2 and AMPK protein. AMPK was added at different concentrations ranging from 7.82 nM to 125 nM and KD values were calculated. n = 3. G Co-IP of SHP2 and AMPK in Hek293T cells co-transfected with plasmids expressing Flag-AMPK and HA-SHP2. n = 3. H-I The Hek293T cells were co-transfected with plasmid encoding wild type SHP2 (HA-SHP2-WT), mutant SHP2 with deletion of PTP (HA-SHP2-ΔPTP) or SH2 (HA-SHP2-ΔSH2), and Flag-AMPK. n = 3. J Western blotting analysis of pT172-AMPK in Hek293T cells co-transfected with plasmid encoding full length SHP2 (HA-SHP2-FL), PTP deletion SHP2 (HA-SHP2-ΔPTP), and Flag-AMPK. n = 3

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  Fig. 8. Activation of AMPK reverses the detrimental effects of SHP2 on cardiac hypertrophy. A The Adeno-Associated Virus 9 (AAV9)-SHP2 or AAV9-Vector was injected into mice through the tail vein (2 × 1011 vg/mouse), and the mice were then administered Ang II via osmotic pump implantation at 0.11 µL/h for 4 weeks. A769662 (30 mg/kg, i.p.) was administered for the last two weeks. B-C Systolic blood pressure and body weight over a 4-week period. n = 6. D Serum Ang II level was detected by ELISA kit. n = 6. E Representative images of M-mode echocardiogram from each group. n = 6. F-G Statistical graphs of EF% and FS%. n = 6. H-I The mRNA levels of Anp and Myh7 in the heart tissues. n = 6. J Representative images of gross heart. n = 6. K The ratios of HW/TL. n = 6. L-M Representative images and quantitative analysis of WGA staining in heart tissues. Scale bar = 100 μm. n = 6. N-O Representative images and quantitative analysis of Masson staining in heart tissues. Scale bar = 100 μm. n = 6. P Immunoblotting analysis of pT172-AMPK and AMPK in heart tissues. n = 6. Q Immunoblotting analysis of SDHA and MTCOI in heart tissues. n = 6. R Analysis of mitochondria score. n = 6. Data are presented as…