cat l5300  (TargetMol)

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Schematic of the 2-step drug screening in HCM cybrids and HCM iPSC-CMs with mutant mtDNA. ( B ) Screening of 41 chemical compounds from the mitochondrial drug bank (all 30 μM in 0.1% DMSO) for MMP analysis in 1 HCM cybrid cell clone with the MT-RNR2 mutation. HCM cybrids treated with 0.1% DMSO (orange) were used as baseline. Values represent the mean ± SEM. n = 3 biological replicates. 2-tailed t test. Top candidates ( P < 0.05) were highlighted in green. ( C ) Analysis of MMP (dark blue) from selected compounds. The MMP of 143B is shown in grey. Con, control. ( D ) Examination of total ATP production in response to DNJ, astragalus polyphenols (Astra), or verbenalin (Verb) administration. Values represent the mean ± SEM. n = 3 biologically independent experiments in 3 lines. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( E ) Relative mitochondrial ATP production was measured using recording buffer (containing 5 mM 2-DG and 5mM pyruvate). Values represent the mean ± SEM. n = 3 biologically independent experiments in 3 lines. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( F ) Galactose-induced cell death assay in HCM iPSC-CMs. Time course (left) and survival rate quantification (right) are shown as the mean ± SEM, n = 3 biologically independent experiments. 2-way ANOVA analysis. *** P < 0.001. Data are representative of 3 independent experiments.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Mutagenesis, Control

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Measurement of MMP analysis. n = 3 biologically independent experiments in 2 cell lines. Values represent the mean ± SEM, 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( B ) Representative concentration-response curves are shown with MMP as an indicator. n = 3 biologically independent experiments. Values represent the mean ± SEM. Data are representative of 3 independent experiments. ( C ) Cardiomyocytes were incubated with indicated concentrations of DNJ for the indicated time periods. Cell growth was determined using a CCK8 assay. n = 3 biologically independent experiments. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. * P < 0.05. ( D ) Analysis of mitochondrial calcium by RHOD-2 indicators in iPSC-CMs. n = 3 biologically independent experiments in 2 cell lines. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. ** P < 0.01, *** P < 0.001.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Concentration Assay, Incubation, CCK-8 Assay

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Schematic identifying the molecular target of DNJ combining pulldown and mass spectrum-proteomics analysis. ( B ) The Coomassie staining gel of eukaryotic purified Flag-His-OPA1 is shown. ( C ) Immunoblot confirmation of the DNJ-binding protein with purified Flag-His-OPA1. ( D ) Immunoblot confirmation of the endogenous DNJ-binding protein in cell lysis using OPA1 antibody. ( E ) MST assay for the affinity between DNJ and purified EGFP-OPA1 protein. ( F ) HCM cybrids were treated with DNJ for 8 hours and collected to purify the mitochondria. Mitochondria were incubated with 10mM EDC for 30 minutes. Proteins were separated by SDS-PAGE and immunoblotted using anti-OPA1 antibodies. ( G ) HCM cybrids treated with DNJ for different time periods (1, 2, 4, 6, and 8 hours). The above mitochondria were then treated as in ( F ). Proteins were separated by SDS-PAGE and immunoblotted using anti-OPA1 antibodies. ( H ) Mitochondria were isolated from cybrids and treated with DNJ or DMSO for 30 minutes. The above mitochondria were then treated as in ( F ). ( I ) Graphical illustration of OPA1 function in mitochondrial cristae remodeling. ( J and K ) Endogenous co-IP assay using IgG and OPA1 antibodies was performed to detect OPA1-IMMT ( J ) and OPA1-ATP5B ( K ) interactions.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Staining, Purification, Western Blot, Binding Assay, Lysis, Incubation, SDS Page, Isolation, Co-Immunoprecipitation Assay

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Mitochondrial networks of control cybrids, HCM cybrids and HCM cybrids with DNJ. Cybrids were immunolabeled for the mitochondrial marker TOM20. Scale bar, 20 μm. ( B ) Representative TEM recordings of cybrids. Scale bar for left images, 2 μm. Scale bar for enlarged images, 200 nm. ( C ) Quantification of the overall cristae morphology on TEM recordings. ( D ) Quantification of mitochondrial cristae number on TEM recordings. Con: n = 47, HCM: n = 61, HCM + DNJ: n = 45 biologically independent mitochondrion. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. ** P < 0.01, *** P < 0.001. ( E ) Diameter of cristae. Con: n = 241, HCM: n = 160, HCM + DNJ: n = 195 biologically independent mitochondrion. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( F ) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the cristae number. Con: n = 47, HCM: n = 61, HCM + DNJ: n = 45 biologically independent mitochondrion. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. ** P < 0.01, *** P < 0.001.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Control, Immunolabeling, Marker

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) The mitochondria isolated from cardiomyocytes were incubated with crosslinker EDC as above. ( B ) Representative TEM recordings. Scale bar, 200 nm. ( C ) Quantification of the overall cristae morphology. ( D – F ) Quantification of mitochondrial cristae number ( D ), Diameter of cristae ( E ) and CJ frequency ( F ). Con: n = 101, HCM: n = 85, HCM + DNJ: n = 92 biologically independent mitochondrion for cristae number and CJ frequency. Con: n = 620, HCM: n = 332, HCM + DNJ: n = 467 biologically independent mitochondrial cristae for cristae diameter. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Isolation, Incubation

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Representative TEM recordings of Con-CMs, HCM-CMs scramble, HCM-CMs scramble + DNJ, siOPA1 and siOPA1 + DNJ. Scale bar, 200 nm. ( B ) Quantification of the overall cristae morphology on TEM recordings. ( C ) Quantification of mitochondrial cristae number. Con: n = 50, HCM scramble: n = 76, HCM scramble + DNJ: n = 69, HCM siOPA1: n = 70, HCM siOPA1 + DNJ: n = 85 biologically independent mitochondrion. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. * P < 0.05, *** P < 0.001. ( D ) Mitochondrial membrane potential was measured. n = 3 biologically independent experiments in 2 cell lines. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( E ) ADP/ATP ratio was measured using a bioluminescent assay system. n = 3 biologically independent experiments in 2 cell lines. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( F ) Analysis of mitochondrial calcium by RHOD-2 indicators in 7 groups. n = 3 biologically independent experiments in 2 cell lines. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Membrane

Journal: The Journal of Clinical Investigation

Article Title: 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy

doi: 10.1172/JCI164660

Figure Lengend Snippet: ( A ) Representative images of H&E staining, wheat germ agglutinin (WGA) staining (cardiac hypertrophy), and Picrosirius red staining (fibrosis). Scale bar, 1,000 μm (HE); 30 μm (WGA); 20 μm (Sirus Red). ( B ) LV collagen volume was assessed and quantified. n = 7 biologically independent samples of cardiac mouse tissues. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( C ) Quantitative results of average cross-sectional areas. n = 7 biologically independent samples of cardiac mouse tissues. Values represent the mean ± SEM. 1-way ANOVA followed by Tukey’s test. *** P < 0.001. ( D ) Immunoblotting ANP protein levels in cardiac tissues. ( E ) Western blotting of mitochondrial OPA1 with EDC treatment in cardiac tissues of Sham, AngII, and AngII + DNJ groups. ( F ) Representative TEM recordings. Scale bar, 500 μm. ( G ) Immunoblotting mitochondrial electron transport chain complex subunits expression. ( H and I ) Cardiac cells were isolated from the cardiac tissues of each mouse, separately. Relative MMP and ATP levels were then measured. n = 5 biologically independent samples of cardiac mouse tissues. 1-way ANOVA followed by Tukey’s test. ** P < 0.01, *** P < 0.001.

Article Snippet: We narrowed down the original mitochondrial targeting compound library (L5300) from TargetMol to 41 chemical compounds by checking their potential to improve mitochondrial fitness from previous references ( ).

Techniques: Staining, Western Blot, Expressing, Isolation