P6281Over expression of HINT2 preserves the mitochondria function of cardiomyocytes via regulating the NAD+ homeostasis

Abstract

Abstract Background HINT2, a member of the histidine triad proteins family, has been proved to positively regulate mitochondrial function and energy metabolism.However, its effect in cardiomyocytes is largely unknown. Purpose To explore the effects of HINT2 on mitochondrial function and ascertain the underlying mechanism in cardiomyocytes Methods The neonatal mouse cardiomyocytes was isolated and cultured for subsequent research. Hypoxia as a adverse factor was used to stimulate the cardiomyocytes for 12 hours. The expression of HINT2 in neonatal mouse cardiomyocytes was determined by real-time quantitative polymerase chain reaction and immunoblotting. We used adenovirus vector to over express or knock down HINT2 in cardiomyocytes.Cell viability were assayed using CCK-8 kit. The mitochondria function was evaluated via detection of oxygen consumption rates and mitochondrial membrane potential. The mitochondrial membrane potential was observed using confocal microscopy after TMRM fluorescence probe labeling. The OCR were measured with a Seahorse Extracellular Flux (XF)-96 Bioanalyzer. The NAD+,as a hydrogen carrier for oxidation respiratory chain, was measured by colorimetric kit.The crystal structure data of HINT2 protein and NAD+ were subjected to rigid body molecular docking using AUTODOCK4. Results The expression of HINT2 was down-regulated in cardiomyocytes after hypoxic stimulus for 12 hours. Over expression of HINT2 protected the cardiomyocytes from hypoxia induced reduced cell viability. Moreover,the mitochondrial membrane potential was higher and the basal, ATP-linked and maximum OCR were increased in HINT2 overexpressing cells under hypoxic stimulus. Conversely,the mitochondrial membrane potential, the basal, ATP-linked and maximum OCR were decreased in cell with HINT2 under expression in normoxia condition. Furthermore,the NAD+ levels was increased in HINT2 overexpressing cells and decreased in cells with knock-down expression of HINT2. Besides,The beneficial effects of HINT2 overexpression can be blocked by nicotinamide phosphoribosyltransferase inhibitor. Moclecular docking results showed that HINT2, as a nucleotide binding protein could docking with NAD+, and the conformation with the strongest binding force has 2 hydrogen bonds which was at ARG99:HH21 and GLN102:HE22 in HINT2. Conclusions Our results suggest that overexpression of HINT2 preserve the mitochondrial function in cardiomyocytes via maintaining NAD+ homeostasis. Acknowledgement/Funding Natural Science Foundation of China