Ethics statement
All animal experiments were carried out according to the guidelines of the Care and Use of Laboratory Animal (NIH Pub. 8th edition, 2011) and authorized by the Animal Experiment Ethics Committee of the Army Medical University (the Third Military Medical University) in Chongqing, China.
Animal studies
The MAP4 (S667A, S737E, and S760E)-KI mice were generated and bred as previously described 17; wild-type (C57BL6/J) mice were purchased from the Animal Centre, Army Medical University (Third Military Medical University, CHN). Male mice, 8-10-weeks-old and weighing 18-22 g, were used for the experiments. The mice were fed a standard rodent chow diet, allowed access to water ad libitum, and housed under 12-hour light/dark cycles. Prior to the experiments, all animals were allowed to acclimate to the facility for one week. The animals were randomly divided into four groups, control, MAP4-KI, hypoxia, and hypoxia+MAP4 KI. Hypoxia was induced with 7.5% O2, 0.1% CO2 and 92.5% N2 for seven days. The desired temperature (22℃) was maintained using a Forma Series II Water Jacket CO2 incubator (model 3131; Thermo Fischer Scientific, Waltham, MA, USA) and the O2 level was controlled through a continuous flow of nitrogen. Afterward, the mice were immediately euthanised, and their heart tissues were collected for transmission electron microscopy or western blot analysis.
Primary cardiomyocytes culture and Hypoxia
Primary cardiomyocytes were isolated according to the conventional methods of our group 11. Firstly, neonatal C57BL6/J or MAP4 KI mice (1-2 days old) were obtained. The hearts were digested with trypsin and collagenase, and then cultured in 6, 24 and 96-well culture plates and maintained for 72 h in Dulbecco's Modified Eagle's Medium (DMEM)-F12 (HyClone) containing 5-bromodeoxyuridine (BrdU; 31mg/L), 10% fetal bovine serum (FBS; Gibco) and penicillin (100U/mL). The primary cardiomyocytes were incubated at 37℃, 5% CO2, and 95% humidity. Hypoxia was induced with 1% O2, 5% CO2, and 94% N2. The desired temperature (37℃) and O2 level were maintained by a Forma Series II Water Jacket CO2 incubator (model: 3131; Thermo Scientific) through a continuous flow of nitrogen.
Mitochondria fractions preparation
Mitochondrial fractions were prepared and validated from primary cardiomyocytes according to the manual instruction of Cell Fractionation Kit (Abcam, ab109719). Briefly, primary cardiomyocytes were collected, counted and diluted to 6.6*10^6 cell/mL. Then, after centrifuged at 500g at 4℃ for 1min, the supernatant was collected and again centrifuged at 10000g at 4℃ for 1min. Then, the pellets were collected and resuspend, and after same centrifugation process, the supernatants containing mitochondrial proteins were collected.
Western blot (WB) analysis
The proteins were extracted as previously reported 11. Briefly, samples were harvested using lysis buffer containing a protease inhibitor, phosphatase inhibitor and PMSF (Beyotime, China). After quantitated with Quick StartTM Bradford 1×Dye Reagent (Bio-Rad, USA), the protein extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA), and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Furthermore, the dissected PVDF membranes were incubated overnight at 4℃ with the following primary antibodies: LC3 (Sigma, 1:2000), Atg3 (CST, 1:1000), Atg7 (CST, 1:1000), Beclin1 (CST, 1:1000), Atg16L1 (CST, 1:1000), Atg5-Atg12 (CST, 1:1000), Bcl-2 (abcam, 1:1000), p62 (Invitrogen, 1:1000), SERCA2 (abcam, 1:1000), Golgin97 (proteintech, 1:1000), HSP60(abcam, 1:1000), Tom70 (Santa Cruz, 1:500), HA tag (proteintech, 1:1000), p-MAP4 (1:1000), MAP4 (Affinity, 1:1000), p-MAP4 (S768) (Biolegend, 1:5000), p-MAP4 (S787) (GL Biochem, 1:1000), p-MAP4 (S737) (GL Biochem, 1:1000), ubiquitin (CST, 1:1000) and β-actin (Proteintech, 1:5000). After successively incubating with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection kit (Amersham Pharmacia, Piscataway, NJ), the PVDF membranes were visualized and quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, United States) and Quantity One software (Bio-Rad), respectively.
Transmission Electron Microscope (TEM)
The procedure of this part was according to previous experiment 17. After fixed in 2.5% glutaraldehyde, primary cardiomyocytes or left ventricle (LV) myocardium were successively subjected to dehydration, vibratome sliced and recut on a microtome and stained with uranyl acetate and lead citrate overnight. The sections were visualized through a TEM (JEM-1400 plus, Japan).
Co-immunoprecipitation
To address the interaction between MAP4/Bcl-2, MAP4/LC3, Bcl-2/Beclin1, and MAP4/Ubiquitin. Cardiomyocytes were lysed in RIPA buffer with a protease inhibitor tablets. The MAP4, Bcl-2, Ubiquitin or LC3 antibodies were incubated with cell lysate for 24 h at 4 °C, then the samples were precipitated with protein A/G-Sepharose (Santa Cruz, Cat# sc-2003) overnight at 4 °C. The precipitates were washed 5 times with PBS at 0 °C and separated by SDS-PAGE and probed by rabbit anti-MAP4 (1:1000, Affinity, USA), anti-LC3 (Sigma, 1:2000), anti-Beclin1 (CST, 1:1000), anti-Bcl-2 (CST, 1:2000) and anti-ubiquitin (CST, 1:1000) antibodies using WB.
Immunofluorescence Staining
The immunofluorescence was performed as previously reported17. Cardiomyocytes were fixed with 1% paraformaldehyde and then permeabilized in 1% Triton X-100 (Sigma, T9284). The nonspecific binding sites were blocked with 10% normal goat serum for 60 min. Cells were stained with p-MAP4 antibody at 1:100, and fluorescence conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1:100 and Alexa Fluor 561-conjugated goat anti-mouse IgG antibody at 1:100. To detect mitochondrial membrane potential, the living cells are stained with TMRE (200nmol/L) for 20min. The cells were viewed and analyzed through a TCS SP5 laser confocal microscope (CANT; Leica, Wetzlar, Germany) or Leica LAS AF 2.3.0 software (Leica Microsystems, Germany), respectively.
RFP-LC3, GFP-LC3, mRFP-GFP-LC3 and Dsred-Mito adenoviruses
The mRFP-GFP-LC3, mRFP-LC3, GFP-LC3 and Dsred-Mito adenovirus were purchased from Hanbio Biotechnology (Shanghai, China) to respectively detect autophagy flux and mitophagosome. The cardiomyocytes grown on glass coverslips in 24-well plates were transferred with these adenoviruses overnight before corresponding treatment, and then the slides were visualized using a confocal microscope (CANT; Leica, Wetzlar, Germany).
HSP60, Tom70 and MARK4 gene silencing with siRNAs
HSP60, Tom70 and MARK4 siRNAs were purchased from GenePharma (Shanghai, China). Cardiomyocytes were transferred with negative control siRNA or HSP60 or Tom70 or MARK4 siRNAs using Lipofectamine 2000 (Nitrogen, USA) according to the manufacturer's protocol. The cells were maintained for 24 h and then used in subsequent experiments.
Bcl-2, MAP4 (Ala), MAP4 (Glu), MAP4 (PJ), UIM-mutated MAP4 (Glu), LIR-mutated MAP4 (Glu) and BH3-mutated MAP4 (Glu) recombinant adenovirus construction and transduction
Recombinant adenovirus vectors were used to overexpress Bcl-2, MAP4 PJ domain (1–693 aa, S737/S760E), UIM-mutated MAP4 (Glu) (L468/S475A and S737/ S760E), LIR-mutated MAP4 (Glu) (Y34/I37A and S737/ S760E), LIR-mutated MAP4 (Glu) (F47/L50A and S737/ S760E), and BH3-mutated MAP4 (Glu) (L83/G87/D88A and S737/S760E). These adenoviruses were constructed by and purchased from the GeneChem Company (CHN). Furthermore, the adenoviruses encoding the MAP4 (Glu) (S737E and S760E) and MAP4 (Ala) (S737A and S760A) were produced as previously described16,17. Cardiomyocytes, cultured in 6, 24 or 96 well plates, were transduced with the corresponding adenoviruses for 48 hours, the transfection efficiency was assessed by WB.
GST pull-down assay
GST pull-down assays were performed by GeneCreate Biological Engineering CO., Ltd (CHN). In brief, the LC3, MAP4 PJ1 (1-170aa), MAP4 PJ2 (398-547aa), MAP4 PJ1 LIR△34,37 or MAP4 PJ2 LIR△47,50 genes were first inserted into PGEX-6P-1 and pet-sumo vectors. The five recombinant plasmids were subsequently expressed in Escherichia coli, followed by the purification of GST-LC3, HIS-MAP4 PJ1, HIS-MAP4 PJ2, HIS- MAP4 PJ1 LIR△34,37, and HIS- MAP4 PJ2 LIR△47,50. The GST pull-down was conducted according to a previous published protocol 32 and measured via western blotting.
Qualitative (shotgun) protein analysis
Cardiomyocytes were lysed in RIPA buffer containing protease inhibitors. The MAP4 antibody was incubated with the cell lysates for 24 hours at 4 °C, and the samples were precipitated with protein A/G-Sepharose (Cat# sc-2003, RRID: AB_10201400; Santa Cruz Biotechnology, USA) overnight at 4 °C. The precipitates, after five washing steps with PBS at 0 °C, were analysed via label-free shotgun proteomics. In brief, sample preparation, protein digestion, in-gel digestion, liquid chromatography-electrospray ionisation tandem MS analysis (Q Exactive) and sequence database searching and data analysis were performed. The MS data was searched against the Uniport database. Intensity-based absolute quantification (iBAQ) with MaxQuant was performed on the identified peptides to quantify the protein abundance.
Statistical analysis
All data are presented was presented using the mean ± SEM. The statistical analysis was performed using SPSS, v. 22.0. The independent-sample t-test and the one-way analysis of variance were applied for comparisons between two groups or more than two groups, respectively. Pearson’s correlation coefficient was calculated using Image J (Fiji) software. P=<0.05 (two tailed) was used as the threshold to define statistical significance.