Fig 2: TRAP1 mutation increased caspase activity in vitro and in vivo and inflammasome activation, resulting in enhanced apoptosis. (A) Sanger sequencing electropherograms of patient genetic variants. (B) Schematic representation of the domain structure of Trap1. Trap1 has three structural domains, an N-terminal domain (NTD) containing mitochondria targeting sequence (MTS) and an ATPase domain, a middle domain (MD) responsible for client binding, and a C-terminal domain (CTD) for dimerization. The arrows indicate the location of the amino acid switch from Glutamate to Glutamine (p.E93Q) and alanine to threonine switch at the position of 64 (p.A64T) in Trap1. (C, D) Western blot of peripheral blood mononuclear cells (PBMCs) from the patient and control was stimulated with LPS and Pam3C or unstimulated (RPMI) for 30 minutes. Cell lysates were run on a 10% SDS gel, transferred to PVDF membrane, and probed for different antibodies. β-actin was used as a loading control. (E) A549 cells were transfected with empty vector, Trap1 wild-type (WT), Trap1 p.A64T (A64T), Trap1 p.E93Q (E93Q), and Trap1 p.A64T+Trap1 p.E93Q (A64T+E93Q) and were stimulated with LPS and Pam3C and the cell lysates were analyzed by Western blot. All Western blots were repeated three times. (F) PCP infection inhibits cell viability in A549 Trap1 transfected cells. The cell viability was measured using the XTT Assay. Values are the mean 3 ± SEM from nine replicates of three independent experiments (n=3). (G) Apoptosis detection by flow cytometry with dual annexin V-PI cell labeling. PBMCs from control (n=7) and patient (n=3) were collected and apoptosis was induced and uninduced by staurosporine. Data is represented in a scatter plot (+ median). Statistical analysis was performed using Student’s t-test for PI- control PBMCs vs PI- patient PBMCs in media, and PI+ control PBMCs vs PI+ patient PBMCs in staurosporine. n=7 for control and n=3 for patient. * p ≤ 0.05. Upper left quadrant (Q1), necrotic cells; upper right quadrant (Q2), late apoptotic or necrotic cells; lower left quadrant (Q3), intact cells; lower right quadrant (Q4) early apoptotic cells. The number in each quadrant refers to the percentage of cells in each subpopulation. The figure is representative of 10,000 cells analyzed independently in seven volunteers and a triplicate of patient cells. RPMI, Roswell Park Memorial Institute Medium; SDS, Sodium dodecyl Sulfate; LPS, lipopolysaccharide; Pam3c, Pam3CysSerLys4; PVDF, polyvinylidene fluoride.

Fig 3: Mitophagy-related biomarkers and TRAP1 downregulation in diabetic cardiomyopathy models. A Screening of DEGs between DCM rats and healthy control rats. B The intersection of MRGs and DEGs is shown in a Venn diagram. C-E Twenty of the most significant gene ontology terms. F Twenty of the most significant KEGG pathways. G Protein–protein interaction network constructed with the DEGs. H HG/PA intervention for 24 h resulted in decreased TRAP1 protein levels in AC16 cells. I IHC tests revealed that the expression of TRAP1 in the myocardial tissue of the diabetic group decreased, scale bar: 500 µm. The data are presented as the means ± SDs. Group comparisons were performed by one-way analysis of variance followed by Tukey’s post hoc test. *p < 0.05. **p < 0.01. ***p < 0.001. ns, not significant

Fig 4: TRAP1 inhibits KANSL3 acetylation to mediate mitophagy of cardiomyocytes under diabetic conditions. A RT‒qPCR was used to measure the mRNA level of KANSL3 in AC16 cells from different groups. B AC16 cells were treated as indicated for 24 h. Western blots of WCLs from AC16 cells were performed with the indicated antibodies. C Western blots of mitochondria isolated from AC16 cells were performed with the indicated antibodies. D Representative images of immunofluorescence staining showing KANSL3 (red) and TOM20 (green) colocalization in AC16 cells. Scale bar: 50 µm. E Immunohistochemical staining of KANSL3 in cardiac tissue sections under different conditions. Scale bar: 250 µm. F Immunoblot analysis of WT AC16 cells overexpressing HA-tagged KANSL3. G Coimmunoprecipitation of HA-KANSL3 and acetylated proteins under HG/PA conditions. H Coimmunoprecipitation of KANSL3 and acetylated proteins in Flag-tagged TRAP1-overexpressing AC16 cells under HG/PA conditions. For comparisons between two groups, statistical significance was determined using a two-tailed unpaired Student’s t test. Group comparisons were performed by one-way analysis of variance followed by Tukey’s post hoc test. *p < 0.05. **p < 0.01. ns, not significant