Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , Metabolism of 13 C 6 ‐glucose. Horizontal columns indicate the fraction labeled in labeled metabolites in the WT and Mybpc3 −/− mice, total labeling of each metabolite normalized to 13 C 6 ‐glucose. B , The experimental design of Seahorse analysis. C , OCR was measured by Seahorse analysis with the indicated reagents in cardiomyocytes slices from Mybpc3‐KO (6‐week‐old) mice with PBS or pyruvate dehydrogenase kinase 4 inhibitor treatment. D , Basal respiration and glucose oxidation dependency were decreased in knockout mice PDK4‐IN‐1 treatment compared with PBS treatment. The glucose oxidation dependency measured by Seahorse XFe24 analyzer as described in the “ ” section. Statistical significance was calculated by Mann–Whitney U test. * P <0.05. Acetyl‐CoA indicates acetyl‐coenzyme A; BPTES, bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide; ETO, etomoxir; KO, knockout; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; OCR, oxygen consumption rate; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and WT, wild type.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Labeling, Knock-Out, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , Immunohistochemical images and statistical analysis of the expression of PDK4 in Mybpc3 −/− mice at 2, 3, and 4 weeks. Scale bars represented 50 μm. B , The immunostaining of ACTN2 and PDK4 in different groups of human patients. C , The experimental design to construct conditional knockout of cardiac Pdk4 in Mybpc3 ‐KO mice. Pdk4 f/f ; Myh6‐Cre mice were generated through the crossbreeding of Pdk f/f mice with Myh6‐Cre transgenic mice. D , The identification of genotypes in different groups. E , Statistical analysis of the body weight of 3 groups. F , The representative short‐axis echocardiography images of different groups and statistical analysis of heart failure and size of left ventricle. G , Representative Masson trichrome staining images illustrating fibrosis in Mybpc3 ‐KO and Pdk4 ‐CKO mice. Statistical analysis shows significant reduction in fibrosis in Pdk4 ‐CKO mice. H , Representative immunofluorescence staining images and quantitative analysis showing the expression levels of ACTN2 and PDK4 in different groups of mice: WT, Mybpc3 −/− , and Mybpc3 −/− / Pdk4 f/f ; Myh6‐Cre ; scale bars represent 20 μm. Data are represented as mean±SD. Statistical significance was calculated by Kruskal–Wallis test with Dunn correction, * P <0.05, ** P <0.01, *** P <0.001. ACTN2 indicates actinin alpha 2; CKO, conditional knockout; EF, ejection fraction; KO, knockout; LVESD, left ventricular end‐systolic diameter; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; Mybpc3 ‐KO/ Pdk4 ‐CKO, Mybpc3 and Pdk4 conditional double knockout mice; ns, not significant; PDK4, pyruvate dehydrogenase kinase 4; Pdk4 ‐CKO, Pdk4 conditional knockout mice; and WT, wild type.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Construct, Knock-Out, Generated, Transgenic Assay, Staining, Immunofluorescence, Double Knockout

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , The experimental design to evaluate the effect of PDK4‐IN‐1 on Mybpc3 ‐KO mice. B , The representative short‐axis echocardiography images of different groups and statistical analysis of heart failure and thickness of left ventricle. C , The heart from 3 groups and statistical analysis of heart weight. D , The representative Masson trichrome staining images and statistical analysis. E , The immunostaining of ACTN2 and PDK4 in different groups of mice (n WT =6, n Mybpc3 ‐KO =7, n Mybpc3 ‐KO+PDK4‐IN‐1 =7). Data are represented as mean±SD. Statistical significance was calculated by Kruskal–Wallis test with Dunn correction, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ACTN2 indicates actinin alpha 2; EF, ejection fraction; KO, knockout; LVESD, left ventricular end‐systolic diameter; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; ns, not significant; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and WT, wild type.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Staining, Immunostaining, Knock-Out

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , Schematic of the experiments procedure in 4 groups of mice: (1) PDK4‐CKO 4W: intragastric administration of PBS in PDK4‐CKO mice once every 2 days from 2W to 4W; (2) Mybpc3 ‐KO 8W+PDK4‐IN‐1: intragastric administration of PDK4‐IN‐1 in Mybpc3‐KO mice once every 2 days from 6W to 8W; (3) Mybpc3‐KO/Pdk4‐CKO 8W: intragastric administration of PBS in Mybpc3 ‐KO/ Pdk4 ‐CKO mice once every 2 days from 6W to 8W; (4) Mybpc3 ‐KO/ Pdk4 ‐CKO 8W+Tamoxifen: intragastric administration of tamoxifen in Mybpc3 ‐KO/ Pdk4 ‐CKO mice once every 2 days from 6W to 8W. B , M mode echocardiography of mice in each group. C , Statistics of EF of each group. D , H&E and Masson trichrome staining of mice in different treated groups (n=3). E , Quantification of ventricular fibrosis. F , Statistics of HW to BW ratio (HW/BW). G , TEM images of cardiac tissue in each group. Data are represented as mean±SD. Statistical significance was calculated by Kruskal–Wallis test with Dunn correction, * P <0.05, ** P <0.01, *** P <0.001. BW indicates body weight; CKO, conditional knockout; EF, ejection fraction; H&E, hematoxylin and eosin; HW, heart weight; ns, not significant; KO, knockout; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; Mybpc3 ‐KO/ Pdk4 ‐CKO, Mybpc3 and Pdk4 conditional double knockout mice; PDK4, pyruvate dehydrogenase kinase 4; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and TEM, transmission electron microscopy.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Staining, Knock-Out, Double Knockout, Transmission Assay, Electron Microscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

doi: 10.1016/j.apsb.2018.10.008

Figure Lengend Snippet: Chrysophanol (CHR) attenuated DOX-induced cardiomyocyte apoptosis and mitochondrial injury in vitro . (A) The chemical structure of CHR (1,8-dihy-droxy-3-methylanthraquinone). (B) H9C2 cells were incubated with the different doses of DOX (0, 1, 5, 25, 50, 100, and 200 μmol/L) for 12 h, and the cell viability were measured by MTS assay. H9C2 cells were pre-cultured with CHR at different doses (1, 10 and 20 μmol/L) for 12 h before stimulation with DOX (1 μmol/L). (C) The cellular morphology was observed by light microscopy. Sclae bar: 200 nm. (D) The nuclear condensation was identified by Hoechst 33342 staining. Sclae bar: 100 nm. (E)–(G) The mitochondrial membrane potential (Δ ψm ) and matrix swelling was determinated using staining with TMRE, Rh123 or Mitotracker. Sclae bar: 100 nm. Representative images of five independent experiments are shown. (H) The cleavage and activation of PARP1 and caspase 3, as well as the BCL-2/BAX ratio were detected by Western blot analysis. (I) The cardiac mitochondria were isolated from H9C2 cells, and the lysed extracts were submitted to Western blot analysis to measure the release of Cyt c from mitochondria to cytoplasm. (J) The cardiac PARylation levels of H9C2 cells were measured by Western blot analysis. The results were normalized to those of VDAC1 or α -tubulin and were presented as the means±SEM. * P < 0.05 vs . the control group, # P < 0.05 vs. the DOX group, n = 3.

Article Snippet: Chrysophanol (CHR, purity 99.02%) was purchased from MCE (MedChemexpress, USA), and was diluted in warm DMSO to 20 mmol/L ( A).

Techniques: In Vitro, Incubation, MTS Assay, Cell Culture, Light Microscopy, Staining, Membrane, Activation Assay, Western Blot, Isolation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

doi: 10.1016/j.apsb.2018.10.008

Figure Lengend Snippet: Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) protected against DOX-induced heart injury in Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A)–(C) Gross hearts, Sirus red and HE-stained transections of the left ventricle. Sclae bar: 100 nm. (D)–(F) The heart weight (HW), the body weight (BW), as well as the heart weight to the tibia length (HW/TL) ratios were calculated. The results were attended as the means ± SEM. * P < 0.05 vs . Normal saline group, # P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Article Snippet: Chrysophanol (CHR, purity 99.02%) was purchased from MCE (MedChemexpress, USA), and was diluted in warm DMSO to 20 mmol/L ( A).

Techniques: Injection, Sterility, Saline, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

doi: 10.1016/j.apsb.2018.10.008

Figure Lengend Snippet: Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) relieved DOX-induced heart dysfunction of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A) and (A׳) The representative echocardiographic graphs are presented. (B)–(I) The echocardiographic parameters were measured, including the ejection fraction (EF), fractional shortening (FS), cardiac output (CO), heart rates (HR), interventricular septum (IVS), left ventricular diameter (LVID), left ventricular volume (LVV) and left ventricular posterior wall thickness (LVPW). The results were attended as the means ± SEM. * P < 0.05 vs . Normal saline group, # P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Article Snippet: Chrysophanol (CHR, purity 99.02%) was purchased from MCE (MedChemexpress, USA), and was diluted in warm DMSO to 20 mmol/L ( A).

Techniques: Injection, Sterility, Saline

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

doi: 10.1016/j.apsb.2018.10.008

Figure Lengend Snippet: Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) relieved DOX-induced cardiac apoptosis of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A) The sections of rat heart were stained with TUNEL labeling, were observed by light microscopy. Representative images of five independent experiments are shown. Sclae bar: 100 nm. (B) The lysed extracts were detected the apoptotic protein levels, such as the protein cleavage and activation of PARP1 or caspase 3, as well as the BCL-2/BAX ratio. The results were presented as the means ± SEM. * P < 0.05 vs . Normal saline group, # P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Article Snippet: Chrysophanol (CHR, purity 99.02%) was purchased from MCE (MedChemexpress, USA), and was diluted in warm DMSO to 20 mmol/L ( A).

Techniques: Injection, Sterility, Saline, Staining, TUNEL Assay, Labeling, Light Microscopy, Activation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

doi: 10.1016/j.apsb.2018.10.008

Figure Lengend Snippet: Chrysophanol (CHR) and 3-aminobenzamide (3AB) both relieved DOX- induced mitochondrial dysfunction of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A)–(E) The rat heart state III and IV respiration, the heart respiratory control ratio (RCR), ADP/O and ATP content was respectively determined. (F) The heart PARylation levels of SD rats were photographed using EVOS FL Auto. Representative images of five independent experiments are shown. * P < 0.05 vs . Normal saline group, # P < 0.05 vs. the DOX group, n =10. ns: no statistical difference.

Article Snippet: Chrysophanol (CHR, purity 99.02%) was purchased from MCE (MedChemexpress, USA), and was diluted in warm DMSO to 20 mmol/L ( A).

Techniques: Injection, Sterility, Saline, Control