Animal experiment were performed in accordance with the NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Airforce Medical University. Leptin receptor-deficient (db/db) and age-matched male littermates (db/+) were provided by Shanghai Model Organisms (Shanghai, China).
Nicotinamide Riboside (NR) was custom synthesized as previously described. Twelve-week-old diabetic db/db and non-diabetic db/+ animals were each given NR via daily oral gavage at a dose of 400mg/kg/d for 4 weeks. The dosage of NR was chosen based on previous studies about the effect of NR on obese mice. At the end of the experiments (when the mice were 16-weeks-old), the hearts of mice were collected.
Primary culture of neonatal rat cardiomyocytes
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Airforce Medical University. Primary neonatal cardiomyocytes were prepared from neonatal rat (0-3 d) hearts as previously described. According to the results of our previous study, HG/HF (25mmol/L glucose and 500umol/L palmitate) medium were employed to induce type 2 diabetes in neonatal primary cardiomyocytes, while HG (25mmol/L) medium were chosen as control medium for all in-vitro experiments in this study. Thereafter, the cardiomyocytes were subjected to normal-glucose medium (25 mmol/L glucose) or high-glucose high-fat medium (25 mmol/L glucose and 500μmol/L palmitate) for 24 hours. For NR treatment, the cells were incubated in NR (2 mmol/L) as previously described.
Echocardiography was performed in M-mode with a Vevo-3100 echocardiography system (Visual sonics Inc. Canada) as previously described. Mice were anesthetized with 2.5% isoflurane. A continuous ECG monitoring system was used to measure mice heart rate of the mice during echocardiography. Left ventricular fractional shortening (LVFS) and ejection fraction (EF) were calculated from the M-mode images using computer algorithms as previously described .
NAD+ content detection
NAD+ levels were measured using a NAD/NADH-Glo Assay Kit (Promega). All procedures were conducted strictly according to the manufacturers’ instructions.
Transmission electronic microscopy (TEM)
Heart sample for TEM detection were prepared according to a procedure previously described. All images were obtained using a transmission electron microscope (JEM-1230, JEOL Ltd, Tokyo, Japan) at 300kV. Mitochondrial images were analyzed by a technician blinded to the treatment using ImageJ software.
Mouse hearts were fixed in 4% paraformaldehyde (PH 7.4) overnight, embedded in paraffin as previously described. Hematoxylin and eosin staining was conducted following standard procedures. Collagen content was detected using Masson trichrome staining.
Immunohistochemistry was performed as previously described. For Mfn2 expression detection in myocardium, primary antibody anti-Mfn2 (1:300, Abcam, USA) were used to incubate with tissue section. At least 10 fields per heart were randomly chosen and analyzed.
Cell apoptosis assay
Terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) assay kit (Roche Applied Science, Swiss) were employed to determine apoptosis in heart tissue. All procedures were conducted strictly according to the manufacturers’ instruction as previously described. A PE-Annexin V Apoptosis detection kit was used to determine cell apoptosis rate before flow cytometry.
Assessment of Mitochondria morphology in cells
Mitochondria morphology was evaluated in primary cardiomyocytes by staining with Mito-tracker Red CMXRos Probe (Thermofisher, USA) as previously described. Images were obtained with a confocal laser-scanning microscope (Nikon A1R MP+ Confocal Microscope, Nikon, Japan). Number and morphology of mitochondria were quantified with ImageJ software as previously described.
Measurement of cellular ROS and Mito-ROS
Cellular ROS (total ROS) and Mito-ROS were determined using staining with a Fluorometric intracellular ROS Kits (Beyotime, China) and Mito-SOX probe (Invitrogen, USA) respectively as previously described. Thereafter, Images were obtained with a confocal laser-scanning microscope (Nikon A1R MP+ Confocal Microscope, Nikon, Japan).
Western-blotting and quantitative real-time PCR
Mice heart and cultured primary cardiomyocytes were lysed with RIPA buffer containing protease inhibitor cocktail. Western blotting analyze were performed as previously described. The primary antibody against the following proteins were used: β-actin (Proteintech, China, #20536-1-AP), Mfn2 (Abcam, #ab58669), Nox4 (Abcam, #ab13303), cleaved-capase3(Cell signaling technology, #9664), PPARα (Novus, #NBP1-04676), PGC1α (Cell signaling technology, #2178).
Total mRNA was extracted from mice heart and primary cardiomyocytes with RNAisoplus (Takara, Japan) and cDNA were synthesized with a PrimerScriptTM RT reagent Kit as previously described. All procedures were conducted following the manufacturers’ protocols. The primer sequences are as shown in table S1.
Adenovirus and siRNA transfection
Primary neonatal cardiomyocytes were transfected with adenovirus harboring Mfn2 shRNA, PPARα, PGC1α, SIRT1. Multiplicity of infection (MOI) was 100:1. For siRNA transfection, Lipofectamine RNAiMAX reagent (Invitrogen) were employed as described previously. Cells were transfected with SIRT1 siRNA, SIRT3 siRNA, PPARα siRNA and PGC1α siRNA following the manufactures’ instructions. After transfected with adenovirus or siRNA, cells were treated with normal medium or high-glucose high-fat medium for 24h.
Chromatin immunoprecipitation (ChIP) assay
CHIP was carried out with a simpleChIP plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) following the protocol provided by the manufacturer as described previously. Sheared chromatins were incubated with a PPARα antibody or a PGC1α antibody. Then the incubated chromatins were fixed with protein G magnetic beads. DNA eluted from the precipitation was detected by PCR analysis. The primers specific to the Mfn2 promoter binding region were as follows: Mfn2 promoter forward: 5’-TGATCCGGAAAGGAAAACAG-3’ and reverse: 5’-CACCGAAAGGCCACAGTAAT-3’.
Luciferase reporter assay
Full-length of 2kb promoter sequence 5’ upstream of the transcription start site of rat Mfn2 were cloned into the PGL3.0-Basic vector upstream of luciferase cassette. For Luciferase reporter assay, a dual-luciferase reporter assay system was used to assess the luciferase activity as previously described. Briefly, HEK-293T cells were transfected with promoter constructs and Renilla luciferase reporter plasmid (Prl-TK). Then the transfected cells were co-infected with PPARα and PGC1α adenovirus or siRNA. The luciferase activity of cell lysis products was measured with a GloMax96 plate reader (Biotek, USA).
Co-IP were performed using a Pierce classic magnetic IP/co-IP kit (Thermo fisher, USA) according to the manufacturers’ instruction as previously described. Lysates samples were incubated with PGC1α antibody or SIRT1 antibody for immunoprecipitation.
All values were presented as Mean ± Standard Error (Mean ± SEM). The statistical difference between two groups was assessed with two-tailed Students’ t test. For groups of three or more, the data were subjects to ANOVA followed by a Bonferroni correction for a post hoc test. A value of P<0.05 were considered as statistically significant difference.