Fig. 6

NHE1 promotes MLKL autophagic-degradation to confer cardioprotection. A Representative image of neonatal rat cardiomyocytes (NRCM) treated with vehicle (Vehi), NaCl (100 mM), LA (25 μM) and Cari (10 μM) during high glucose (35 mM)-oxygen deprivation (HG+OD), oxygen–glucose deprivation (OGD) stimulation and visualized using propidium iodide (PI). Scale bars, 100 μm. B Quantification of PI-positive cells in the total cell area (top, n = 5 for OGD, n = 3 for HG + OD) and cell viability measured by LDH assay (bottom, n = 3 per group) in NRCM treated with vehi, LA, NaCl, and Cari under HG + OD (24 h) and OGD (12 h) conditions. C Representative image and quantitative analysis of PI-positive cells in the total cell area (n = 6) and cell viability (n = 4) in NRCM following treatment with vehi, LA (50 μM), NaCl (100 mM), and Cari (10 μM) under TSZ (TNF-α, 50 ng/mL; SM-164, 5 μM and z-VAD, 25 μM) for 3 h in NRCM. D Western blot analysis of MLKL in NRCM treated with Saline and 100 mM NaCl during OGD (left) and HG + OD (right). E Western blot analysis of MLKL in L929 cells treated with 100 mM and 400 mM NaCl (left) for 30 min or with 100 mM NaCl during TSZ (right) for 60 min. Blots are representative of three independent experiments. F and G Representative images and quantification of PI⁺ cells in the total cell area and cell viability of NRCM infected with Lenti-shNHE1 and treated with TSZ for 3 h, n = 3, scale bars, 100 μm. H The ratio of intracellular pH to LDH release levels was measured by BCECF-AM (3 μM) and CellTiter-Glo assay, n = 4 per group. I Western blot analysis of MLKL in NHE1 overexpression and knockout in the cardiomyocytes with stable constructs, blots are representative of four independent experiments. J Transcriptional levels of MLKL in NRCM subjected to HG + OD for 6 and 12 h were compared between the Saline and 100 mM NaCl treatment groups, n = 3 per group. K and L Western blotting and quantification of MLKL were performed in MLKL-overexpressing cardiomyocytes treated with vehicle, 100 mM NaCl and then treated with 3-Methyladenine (3-MA, 10 mM, 12 h), Chloroquine (CQ, 25 μM, 12 h), Bafilomycin A1 (BafA1, 100 nM, 12 h), Rapamycin (RAPA, 5 μM, 24 h) and MG132 (20 μM, 8 h). Blots are representative of three independent experiments. Statistical significance was determined by One-way ANOVA with Tukey’s test. All quantitative data are expressed as mean ± SD