Fig 2: Structure-based screening of small-molecule activators of SIRT3 and identifies 2-APQC activates SIRT3 deacetylase activity in H9c2 cells. a The structure of SIRT3 and virtual screening scheme model for the discovery of SIRT3 activator. Created with biorender.com and https://smart.servier.com. b The 5 candidate compounds based on virtual screening and molecular docking. c The relative SIRT3 deacetylation activity after the candidate compounds (1, 10, 100 μM) and Honokiol (Hon) (10 μM) treatment. Data are present as mean ± s.e.m, n = 3. d The relative SIRT3 deacetylation activity after Hon or 2-APQC (0.01, 0.1 1, 10, 100 μM) treatment. Data are present as mean ± s.e.m, n = 3. e H9c2 cells were treated with indicated concentration of 2-APQC for 24 h, and cell viability was measured by the MTT assay. Data are present as mean ± s.e.m, n = 3. f H9c2 cells were treated with indicated concentration of 2-APQC for 24 h, and then treated with isoproterenol (ISO) for 48 h, the cell viability was measured by the MTT assay. Data are present as mean ± s.e.m, n = 3. g The molecular docking sketch map to indicate the binding mode between SIRT3 and 2-APQC. h Surface plasmon resonance assay detected the Kd value of SIRT3 in H9c2 cells treated with 2-APQC. i Cellular thermal shift assay detected the thermal stability of SIRT3 in H9c2 cells treated with 2-APQC. j Western blot analysis of SIRT3, acetylated-lysine, acetylated manganese superoxide dismutase 2 (MnSOD2) at K68 and K122 protein expression in H9c2 cells treated with 2-APQC for 24 h. k Western blot analysis of acetylated-lysine, acetylated MnSOD2 at K68 and K122 protein expression in H9c2 cells treated with 2-APQC for 24 h, together with or without ISO/nicotinamide (Nico) as the research design. β-actin, loading control. ns no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Hon Honokiol, Nico nicotinamide, ISO isoproterenol

Fig 3: Transcriptomics-based identification of possible molecular mechanisms of HF improvement effect of 2-APQC in H9c2 cells. a, b The volcano plots showing the differential expression of genes in the heart tissues of CON VS ISO and ISO VS ISO + 2-APQC as shown. c, d Heat map analysis of genes differentially expressed in rat hearts of Con and ISO and ISO and ISO ISO + 2-APQC, grouping all related genes into hierarchical clusters. e, f GO analysis of genes differentially expressed in rat hearts of Con and ISO and ISO and ISO ISO + 2-APQC. g SIRT3-regulated PPI after 2-APQC treatment

Fig 4: 2-APQC alleviates myocardial fibrosis to improve HF by SIRT3 activation in vivo. a, b Immunohistochemical detection of ac-MnSOD2 (K68) and ac-MnSOD2 (K122) in rat heart tissues in different groups. Data are present as mean ± s.e.m, n = 3. c The expression levels of Ac-MnSOD2 (K68), ac-MnSOD2 (K122), SIRT3 and acetylated lysine were detected by Western blot. d, f Immunohistochemical detection of α-SMA cells with α-SMA in rat heart tissues in different groups. The position of the nucleus is marked by DAPI staining. Scale bar = 25 μm. Data are present as mean±s.e.m, n = 3. e, g Immunohistochemical detection of masson staining in rat heart tissues in different groups. Data are present as mean±s.e.m, n = 3. h Detection of Akt/ mTOR/p70S6K pathway by Western blot. i Detection and quantification of the expression of TGF-β/Smad3 pathway, TGF-β, JNK, p-JNK, Smad3, p-Smad3 and lysyl oxidase (Lox) by Western blot. j The expression levels of Akt, p-Akt, JNK, p-JNK, α-SMA, fibronectin and collagen I in rat heart tissues in different groups were detected by Western blot. k H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of nicotinamide for 24 h, and then treated with ISO for 48 h, the cell viability was measured by the MTT assay. l H9c2 cells were treated with indicated concentration of 2-APQC in the presence of absence of nicotinamide for 24 h, and then treated with ISO for 48 h. Determine the size of cardiomyocytes by β-actin tracker staining (red). The position of the nucleus is marked by DAPI staining. Scale bar = 10 μm. 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 indicated concentration of 2-APQC for 24 h, and then treated with ISO for 48 h. Determine the size of cardiomyocytes by β-actin tracker staining (green). The position of the nucleus is marked by DAPI staining. Scale bar = 10 μm. Data are present as mean ± s.e.m, n = 3. n Detect the expression of p-AKT, p-mTOR, p-JNK and p-SMAD3 by Western blot. o Detect the expression of p-AKT, p-mTOR, p-JNK and p-SMAD3 by Western blot in SIRT3 KO mice. p Detect the expression of p-AKT, p-mTOR, TGF-β and p-SMAD3 by Western blot. q Detect the expression of JNK, p-JNK, α-SMA, fibronectin and collagen I by Western blot. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with ISO group; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, compared with control group. Nico nicotinamide, ISO isoproterenol

Fig 5: 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