fig7

Figure 7. Impact of S100A13 inhibition/silencing on cardiomyocyte function and intracellular labile Cu⁺, co-IP, and the structure-based protein interaction interface between RAGE and S100A13. (A-F) Effect of RAGE inhibitor FPS-ZM1, cuproptosis inhibitor TTM and S100A13 inhibitor amlexanox (AMX) on MG-AGE-evoked cardiomyocyte defect in vitro. Freshly isolated C57BL/6 mouse cardiomyocytes were challenged with 5 µM MG-AGE for 12 h with or without 25 μM FPS-ZM1, 20 μM TTM, or 20 μM AMX prior to evaluation of cardiomyocyte mechanics. (A) Resting cell length; (B) Peak shortening normalized to cell length; (C) +dL/dt; (D) -dL/dt; (E) TPS; (F) TR₉₀; (G and H) Representative images and pooled summary exhibiting effect of S100A13 silencing on AMX-induced response against MG-AGE-evoked rise in intracellular labile Cu⁺. HL-1 cells transfected with scramble or S100A13 siRNA were exposed to MG-AGE (5 µM) for 12 h in the absence or presence of AMX (20 μM) or FPS-ZM1 (25 μM) prior to assessment of labile Cu⁺ levels; (I) Validation of S100A13 silencing (using 2 batches of plasmids); (J) Co-IP analysis between RAGE and S100A13 in myocardial tissues from C57BL/6 mice; (K) Predicted interaction interface between RAGE and S100A13. Structural representation of the predicted RAGE-S100A13 complex. The RAGE and S100A13 subunits are depicted in cyan and orange, respectively. Interface residues involved in the interaction are highlighted in blue (RAGE) and red (S100A13) to delineate the binding pocket. Mean ± SEM, n = 16 cardiomyocytes from 3 mice (A-F) or 7 independent HL-1 cell cultures (H) per group, normal distribution was determined via the Shapiro-Wilk test, results were evaluated via two-way ANOVA and then Tukey’s post hoc test, ^*P < 0.05 between indicated groups. RAGE: Receptor for AGEs; Co-IP: co-immunoprecipitation; S100A13: S100 calcium-binding protein A13; TTM: tetrathiomolybdate; AMX: amlexanox; MG-AGE: methylglyoxal-derived advanced glycation end product; TPS: time-to-peak shortening; TR₉₀: time-to-90% relengthening; SEM: standard error of the mean; ANOVA: analysis of variance; IgG: immunoglobin G; LEU: leucine; GLN: glutamine; LYS: lysine; ARG: arginine; PRO: proline; THR: threonine; GLU: glutamate/glutamic acid; VAL: valine.