Palmitic acid-evoked PKR/JNK activation and SIRT1 decline: Association with myocardial ageing phenotype changes and partial reversal by inhibitors

Objective: This preliminary study examines possible associations between palmitic acid (PA) exposure and ageing-associated features in cardiomyocytes.

Methods: Cells were treated with PA at0.08-0.8 mmol/L concentrations or for different duration (12-72 h). Cell viability was assessed by using the MTT assay, Cell cycle was analyzed by flow cytometry, and ROS levels were measured by using a ROS kit. We performed SA-β-gal staining to evaluate SA-β-gal-positive cells rate. Concentrations of the senescence-associated secretory phenotype (SASP)-related factors (IL-1A, IL-6, IL-8, and IFN-γ) were measured using ELISA. We used the Whole-transcriptome RNA Sequencing to identify differentially expressed genes (DEGs). Involvement of the PKR/JNK pathway was examined using specific inhibitors (PKR-IN-C16 and SP600125). Western blotting was used to analyze the protein expression of p-PKR, PKR, p-JNK, JNK, SIRT1, p16^INK4a,p-Rb, Rb, IL-1A, and IFN-γ. SIRT1 activity was detected by using a kit.

Results: The cell viability (%) decreased in a dose- and time-dependent manner with PA treatment. After 0.19 mmol/L PA exposure for 12-36 h, cells proportions of G1 phase increased, while that of S phase decreased. ROS levels; SA-β-Gal-positive cell rates; and IL-1A, IL-6, IL-8, and IFN-γ concentrations increased. In0.19 mmol/L and 36 h PA groups, p-PKR, p-JNK, p16^INK4a, IL-1A and IFN-γ expression increased, while p-Rb expression decreased. SIRT1 activity was reduced following PA stimulation. RNA Sequencing identified DEGs involved in ageing-related pathways, with SIRT1 as a key gene module. P-PKR, p-JNK, p16^INK4a, IL-1A, IFN-γ and SA-β-Gal protein expression was lower, while p-Rb expression was higher, inPA+PKR-IN-C16 group than in PA group. P-JNK, p16^INK4a, IL-1A, and IFN-γ protein expression was lower, while p-PKR, SIRT1,p-Rb expression was higher, inPA+SP600125 group than in PA group. SIRT1 activity was higher inPA+PKR-IN-C16 and PA+SP600125groups than in PA group.

Conclusion: Our inhibitor data tentatively suggest that PKR/JNK pathway may contribute-at least in part-to the decline in SIRT1 expression and activity observed during PA-induced myocardial ageing phenotype changes, although a definitive causal link remains to be established.

Myocardial aging phenotype changes; PKR/JNK; Palmitic acid; SIRT1.