Histidine triad nucleotide-binding protein 2 protects cardiomyocytes via maintaining NAD homeostasis in virto and in vivo
Authors: Mengkang F , Huang Y.I.N , Qian J 
Published: November 2020

Abstract

Background
Heart failure (HF) is the end-stage of most heart diseases with poor clinical outcomes. The mitochondrial dysfunction is a critical therapeutic target in HF, and the histidine triad nucleotide-binding (HINT2) protein has been shown to enhance energy metabolism in liver. However, the role of HINT2 in HF remains unclear.

Purpose
To explore the role of the histidine triad nucleotide-binding 2 (HINT2) protein in heart failure.

Methods
Neonatal mouse ventricle myocytes (NMVMs) and myocardial infarction-induced heart failure mice were used for in vitro or in vivo experiments. Adenovirus (ADV) and adeno-associated virus serum type 9 (AAV9) vectors were used to regulate HINT2 expression. The expression of HINT2 was determined by quantifying the mRNA and protein levels. Cell survival was analysed using the CCK-8 kit and TUNEL staining. Mitochondrial function was determined by the mitochondrial membrane potential and oxygen consumption rates. AAV9-HINT2 was injected 24 h post myocardial infarction following which transthoracic echocardiography and histological analyses were performed after 4 weeks. Positron emission tomography tomography-computed tomography (PET/CT) and targeted metabolomics analyses were used to explore the metabolic status in vivo. NAD levels were measured using a colorimetric kit. Computer-simulated rigid body molecular docking was performed using AUTODOCK4. Molecule binding kinetics assays were performed using biolayer interferometry.

Results
After 12 hours hypoxia stimuli, HINT2 was down regulated. ADV-HINT2 induced HINT2 overexpression improved NMVMs survival and reduced apoptosis after hypoxia. MMP was reduced in ADV-shHINT2 group and was preserved in ADV-HINT2 group under hypoxia. HINT2 overexpressed NMVMs showed less reduction in basal, ATP-linked and maximum OCR after hypoxia stimuli. In vivo experiment, showed that cardiac function and metabolic status was preserved by HINT2 overexpression. PET/CT displayed glucose uptake ability was significantly reduced in in failing heart, which was preserved by overexpression of HINT2. Targeted metabolomics analysis showed that nicotinate and nicotinamide metabolism pathway was regulated by HINT2, in which oxidized state NAD (NAD+) and redox state NAD (NADH) was increased in AAV9-HINT2 group. NAD concentration was detected in NMVMs. HINT2 can improve total NAD level rather than ratio of NAD+/NADH, and its effect was limited into mitochondria.HINT2 overexpression restored mitochondrial NAD levels; this was dependent on nicotinamide mononucleotide (NMN). Using computer-simulated molecular docking analysis and biolayer interferometry, we observed that HINT2 potentially binds and associates with NMN.

Conclusion
In summary, these findings demonstrate that the HINT2 is beneficial to preserve the heart function and metabolism in the HF murine model after acute MI, and this positive effect may due to the maintenance of mitochondrial NAD homeostasis.

Funding Acknowledgement
Type of funding source: Foundation. Main funding source(s): National Natural Science Foundation of China (Grant Nos: 81970295, 81870267)