Fig. 6
The SIRT3 activator 2-APQC plays a protective role in ISO-induced heart injury by SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) regulated mitochondrial oxidative stress and SIRT3-AMPK-receptor interacting protein kinase 3 (RIPK3)-modulated necrosis. a H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The expression of PYCR1 was analyzed by western blot. β-actin was measured as a load control. The quantification of western blotting analysis is shown. b The content of reactive oxygen species (ROS) was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. c The expression of p38MAPK and p-p38MAPK was analyzed by western blotting. β-actin was measured as a load control. d H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of proline for 24 h, and then treated with ISO for 48 h. The content of ROS was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. e H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of proline for 24 h, and then treated with ISO for 48 h. The expression of PYCR1 and p-p38MAPK was analyzed by western blotting. β-actin was measured as a load control. f PYCR1 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The content of ROS was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. g PYCR1 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The expression of PYCR1 and p-p38MAPK was analyzed by western blotting. β-actin was measured as a load control. h The content of ROS was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. i H9c2 cells were treated with the specified concentration of 2-APQC and nicotinamide for 24 h, and then treated with ISO for 48 h, western blot analysis of p38MAPK and p-p38MAPK expressions. β-actin was measured as a load control. j SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The expression of PYCR1 and p-p38MAPK was analyzed by western blotting. β-actin was measured as a load control. k SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The content of ROS was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. l SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of proline for 24 h, and then treated with ISO for 48 h. Detect the proline content levels of each group, the quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. m SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The content of ROS was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. n SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of proline for 24 h, and then treated with ISO for 48 h. The expression of SIRT3 and p-p38MAPK was analyzed by western blotting. β-actin was measured as a load control. o The content of Ca2+ was measured by flow cytometry. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. p H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. Flow cytometry was used to detect the level of cell necrosis after PI staining. The quantification of analysis is shown. Data are present as mean ± s.e.m, n = 3. q The expression of AMPK, p-AMPK, Parkin, RIPK3, Caspase-1, Caspase-8 was analyzed by western blotting. r SIRT3 knockdown plasmid transfection was conducted 48 h prior to cell treatment. H9c2 cells were treated with the specified concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. The expression of p-AMPK, Parkin, RIPK3, Caspase 1, Caspase 8 was analyzed by western blotting. β-actin was measured as a load control. s 2-APQC, a small-molecule activator of SIRT3, alleviates heart failure by regulating SIRT3-mediated mitochondrial proline and ROS metabolic homeostasis. Created with biorender.com and https://smart.servier.com/. Data are expressed as mean ± SEM. All data represent at least three independent experiments. ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with control group; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, compared with ISO group. ISO isoproterenol
