Galangin 3-methyl ether alleviates mouse hypertrophic cardiomyopathy via targeting HDAC2 and subsequently inactivating the PI3K-AKT signaling pathway

Figures

• 

  Fig. 1. Galangin 3-methyl ether (G3-ME) treatment alleviates angiotensin II (Ang II)-induced cardiomyocyte hypertrophy in H9c2 cells. a Chemical structural formula of G3-ME. b The toxic effects of G3-ME in H9c2 were determined using the CCK-8 assay. n = 6 per group. c The mRNA expression levels of pathological cardiac hypertrophy marker genes Nppa and Nppb. n = 3 per group. d The protein expression levels of ANP and BNP. n = 4 per group. e Representative images of H9c2 cell morphology were obtained through immunofluorescent staining of cTNT (green), and cardiomyocyte size was analyzed from 10 confocal dishes, with all cardiomyocytes in the different groups being 135, 160, 97, and 153, respectively. The values are mean ± SEM. *P < 0.05 vs. control and #P < 0.05 vs. Ang II in (c). *P < 0.05 in (d, e).

• 

  Fig. 2. Galangin 3-methyl ether (G3-ME) ameliorates angiotensin II (Ang II)-induced human cardiomyocyte hypertrophy. a Schematic diagram of the induced differentiation of human embryonic stem cells (hESC) into cardiomyocytes (hESC-CMs). b The toxic effects of G3-ME in hESC-CMs were evaluated using the CCK-8 assay. n = 6 per group. c The protein expression levels of ANP and BNP. n = 5 per group. d The mRNA expression levels of NPPA and NPPB. n = 4 per group. e Representative images of hESC-CM morphology were obtained through immunofluorescent staining of cTNT (green), and cardiomyocyte size was analyzed from 10 confocal dishes, with all cardiomyocytes in the different groups being 34, 39, 40, and 44, respectively. f Representative images of calcium (Ca2+) transients were recorded in single cells, and Ca2+ transient amplitude, time to peak, peak to peak, and transient duration 50% in hESC-CMs were analyzed from 6 confocal dishes, with all transient cycles in the different groups being 24, 24, 32, and 25, respectively. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 3. Galangin 3-methyl ether (G3-ME) supplementation restores both cardiac systolic and diastolic function in Myh6R404Q mice with pathological cardiac hypertrophy. a Schematic diagram of the mice experimental procedure. b Representative images of gross heart morphology. c The ratio of heart weight (HW) to tibial length (TL). d Representative images of echocardiograms. e The left ventricular (LV) mass. f Echocardiographic analysis of LV anterior wall (LVAW) or posterior wall (LVPW) thickness in systolic and diastolic periods. g Echocardiographic analysis of LV diameter (LVD). h Echocardiographic analysis of LV volume (LVV). i Echocardiographic analysis of LV ejection fraction. j The ratio of E peak to A peak (E/A) of mice. n = 6 per group. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 4. Galangin 3-methyl ether (G3-ME) improves cardiac injury and remodeling in Myh6R404Q mice. a, b The protein expression levels of ANP and BNP in cardiac tissues. n = 4 per group. c The serum NT-proBNP levels. n = 5 per group. d, e The mRNA expression levels of Nppa, Nppb, Acta2, Col1a1, and Col3a1 in cardiac tissues. n = 4 per group. f Representative images of cardiac H&E staining. g The cardiac cross-sectional area determined by H&E staining. n = 6 per group. h Representative images of the cardiac fibrosis area using Masson’s staining. i Quantification of fibrosis area by Masson’s staining. n = 6 per group. j Representative images of cardiomyocytes by WGA staining. k Analysis of cardiomyocyte size using WGA staining from 6 mice, with all cardiomyocytes in the same area analyzed in each section. n = 375, 341, 328, 283, 353, and 340, respectively. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 5. Galangin 3-methyl ether (G3-ME) improves both cardiac function and structure in Tnnt2R109Q mice with pathological cardiac hypertrophy. a Schematic diagram of the mice experimental procedure. b The ratio of heart weight (HW) to tibial length (TL). c Representative images of echocardiograms. d Echocardiographic analysis of left ventricular (LV) anterior wall (LVAW) or posterior wall (LVPW) thickness in systolic and diastolic periods, and the ratio of E peak to A peak (E/A). n = 6 per group. e The serum NT-proBNP levels. n = 5 per group. f Representative images of cardiac H&E staining. g The cardiac cross-sectional area determined by H&E staining. n = 5 per group. h Representative images of the cardiac fibrosis area using Masson’s staining. i Quantification of fibrosis area by Masson’s staining. n = 5 per group. j Representative images of cardiomyocytes by WGA staining. k Analysis of cardiomyocyte size using WGA staining from 6 mice, with all cardiomyocytes in the same area analyzed in each section. n = 332, 348, 292, and 319, respectively. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 6. Galangin 3-methyl ether (G3-ME) inhibits pathological cardiac hypertrophy by regulating the PI3K-AKT signaling pathways. a Schematic diagram for exploring the key targets and signaling pathways of G3-ME. b Principal component analysis (PCA) of the angiotensin II (Ang II)-treated and the G3-ME + Ang II-treated RNA sequence. c Heatmap of differential genes. d KEGG enrichment analysis according to differential genes. e–i The protein expression levels of p-P85, P85, p-AKT, and AKT in H9c2, hESC-CMs, and cardiac tissues of wild-type (WT) or Myh6R404Q mice. n = 4 per group. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 7. Galangin 3-methyl ether (G3-ME) prevents pathological cardiac hypertrophy by directly targeting HDAC2. a The flowchart of network pharmacology and the protein-protein interaction diagram. b KEGG enrichment analysis according to 42 potential targets of G3-ME. c The binding energy between G3-ME and potential targets enriched in the PI3K-AKT signaling pathway. d Representative schematic diagram of molecular docking between HDAC2 and G3-ME. e Schematic diagram of HDAC2 regulating the PI3K-AKT signaling pathway. f, g The protein expression levels of p-HDAC2 and HDAC2 in H9c2, hESC-CMs and cardiac tissues of wild-type (WT) or Myh6R404Q mice. n = 4 per group. h Drug affinity reaction target stability (DARTs) assay was performed in vitro and in vivo on H9c2. i DARTs assay was performed in vitro and in vivo on hESC-CMs. j Cellular thermal shift assay was performed on H9c2 and hESC-CMs, respectively. k The results of surface plasmon resonance indicated a direct binding interaction between G3-ME and HDAC2, with a binding affinity of 3.36 μM. The values are mean ± SEM. *P < 0.05.

• 

  Fig. 8. Interference with HDAC2 expression influences the cardioprotective effect of galangin 3-methyl ether (G3-ME) on PI3K-AKT signaling and cardiac hypertrophy. a The efficiency of HDAC2 knockdown (HDAC2-KD) in H9c2. b, c The mRNA expression levels of Nppa and Nppb in normal control (NC) or HDAC2-KD cells. n = 4 per group. d Representative images of NC or HDAC2-KD cell morphology were obtained through immunofluorescent staining of cTNT (red). e The cardiomyocyte size was analyzed from 8 confocal dishes, with all cardiomyocytes in the different groups being 95, 106, 101, 101, 102, and 95, respectively. f The protein expression levels of p-P85, P85, p-AKT and AKT in NC or HDAC2-KD cells. g The efficiency of HDAC2 overexpression (HDAC2-OE) in H9c2 cells. h, i The mRNA expression levels of Nppa and Nppb in control vector (Ctrl) or HDAC2-OE cells. n = 4 per group. j Representative images of Ctrl or HDAC2-OE cell morphology were obtained through immunofluorescent staining of cTNT (green). k The cardiomyocyte size was analyzed from 8 confocal dishes, with all cardiomyocytes in the different groups being 98, 84, 93, 81, 76, and 77, respectively. l The protein expression levels of p-P85, P85,…

• 

  Fig. 9. Galangin 3-methyl ether (G3-ME) alleviates pathological cardiac hypertrophy both in vivo and in vitro by targeting HDAC2 and subsequently deactivating the PI3K-AKT signaling pathway.