Human heart samples
The study conforms to the principles that govern the use of human tissues outlined in the Declaration of Helsinki. Approval was obtained from the human ethics committee of First Affiliated Hospital of Sun Yat-sen University. All patients or the family of prospective heart donors gave written informed consent prior to participation. Failing human heart samples were collected from 9 patients undergoing heart transplantation because of end-stage heart failure (table S1). Three non-failing control heart samples were obtained from prospective multi-organ donors, which did not exhibit cardiovascular pathology but were unable to be transplanted due to technical reasons. Tissue samples were collected at the time of explantation and rapidly frozen in liquid nitrogen or fixed with 4 % paraformaldehyde.
Animal Studies
Snx3-floxed mice were constructed in the Shanghai Model Organisms Center by CRISPR/Cas9 technology. Briefly, the donor vector containing four guide RNAs and Cas9 mRNA targeting Snx3 introns 2 and 3 was microinjected into C57BL/6 mouse fertilized eggs. The positive founder mice were backcrossed with wild-type C57BL/6 mice to obtain heterozygous Snx3flox/+ mice with germ line transmission. Snx3flox/+ mice were self-crossed to generate homozygous Snx3flox/flox mice. Snx3flox/flox mice were crossed with C57BL/6J-Myh6em1(IRES-Cre)Smoc mice (Shanghai Model Organisms Center, Stock No: NM-KI-00083. MGI ID: 97255) to generate Myh6-Cre+; Snx3flox/flox mice, ie. cardiac-specific Snx3-cKO mice.
Snx3 transgenic mice were constructed in the Shanghai Model Organisms Center using standard methods. Briefly, the Piggybac vector harboring mouse SNX3 cDNA was microinjected into the fertilized egg of C57BL/6 mouse, and the transgenic founder mice were obtained. 3 generations of backcross between each Snx3 transgenic mice and wild-type (C57BL/6) mice were adopted to breed two independent Snx3 transgenic lines. The two transgenic lines were respectively crossed with B6.FVB-Tg(Myh6-Cre)2182Mds/J mice (Jackson Laboratory (Bar Harbor, ME), Stock No: 011038. MGI ID: 2386742.) to generate cardiac-specific overexpressed Snx3 (Snx3-cTg) mice.
The mice were genotyped by PCR and further confirmed by western blot analysis (table S2-S4). All animal protocols were conducted under the institutional guidelines of Animal Care and Use Committee, and were approved by the Research Ethics Committee, Sun Yat-sen University. Experimental animals were housed, bred, and maintained in the specific pathogen-free (SPF) facility of the Experimental Animal Center of Sun Yat-sen University. As we previously described30, the transthoracic 2D-guided M-mode echocardiography (such as heart function and global cardiac volumes) was assessed using by a Technos MPX ultrasound system (ESAOTE, SpAESAOTE SpA, Italy) equipped with an 40-MHz scan probe. The Vevo 2100 imaging software was used for measurements and calculations. Then, the treated mice were sacrificed, and hearts were rapidly sampled for further experiments.
Bioluminescence Imaging47
XenoLightTM D-luciferin potassisum salt (PerkinElmer, P/N 122799) was diluted in sterile PBS to 15 mg/mL, and was respectively injected into the luciferase-labeled Snx3-cTg mice or N-Tg mice (150 mg/kg). A few minutes after injection, mice were anesthetized using 2% isoflurane inhalation (with a 2 L/min oxygen flow rate), and were placed inside the camera box of the IVIS Lumina XR small animal optical imaging system (PerkinElmer). The sequential images of the mice were run every two minutes. Lumazone Version 2.0 software was used to analyze the intensity of the fluorescence (intensity/second) in mice.
Histological Analysis
Myocardial tissue samples were fixed in 10% paraformaldehyde and embedded in paraffin for sectioning. Sections were stained with wheat germ agglutinin (WGA), hematoxylin-eosin (HE), Masson’s and picric sirius red (PSR) for histopathological examination under a light microscope.
For immunofluorescent (IF), paraffin sections of myocardial samples were treated with primary anti-SNX3 (Proteintech, #10772-1-AP) or anti-p-STAT3 (Y705, Cell Signaling Technology, CST, Beverly, MA, USA, #9145) overnight at 4°C. Then, the samples were incubated with CoraLite488/594-conjugated anti-rabbit IgG (diluted 1:200, Proteintech, #SA00013-6 or #SA00013-8), were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI, CST, #4083). Fluorescence was captured by EVOS FL Auto (Life Technologies, Bothell, WA, USA).
Plasmid, recombinant adenoviral vectors and recombinant protein
Vps26 (NM_001007740.1), Vps35 (NM_001105718.2), Snx3 (NM_001044283.1) and Stat3 (NM_012747.2) were constructed by ligating respective full-length cDNA into pcDNA3.1 (+) with Flag or HA-tag. Similarly, short hairpin (sh) RNA targeting the rats importin α3 gene (also called as Kpna3, NM_001014792) (sh-importin α3), the rats Snx3 gene (sh-SNX3) and non-targeting control shRNA (sh-NC) sequences (table S5 and S6) were respectively subcloned into pcDNA3.1 (+) with Flag-tag.
Recombinant adenoviral vectors expressing rats Snx3 cDNA (Ad-SNX3, with Flag-tag), rats STAT3 cDNA (Ad-STAT3, with HA-tag), sh-SNX3 sequence (Ad-sh-SNX3, with Flag-tag) and control vectors were generated by standard procedures. Briefly, the pAdTrack plasmids containing corresponding genes were linearized by using restriction endonuclease PmeⅠ, and were transformed into Escherichia coli strain BJ5183 cells carrying the Ad-Easy-1 plasmid. The successful recombinant plasmids were digested with restriction endonuclease PacⅠ, and were transfected into HEK293A cells to generate Ad-SNX3, Ad-STAT3 or Ad-sh-SNX3. The vectors were purified by plaque, cultured in a large scale, and purified by CsCl step- and isopycnic-gradient centrifugation.
GST-STAT3, GST-SNX3, GST-VPS35 and GST-VPS26 protein were respectively expressed in E. coli BL21 (DE3) maintained in luria-bertani (LB) medium (including 50 ug/mL ampicillin) at 37℃. By the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mmol/L), protein expression was induced at 16℃ overnight to an OD600 value of 0.8. Cells were re-suspended in 20 mmol/L Tris + 200 mmol/L NaCl + 1 mmol/L PMSF, were broken using ultrasonic instrument in ice, and were collected supernatant after centrifugation (1,2000 rpm for 3 h at 4°C). These recombinant protein were purified by GST-Sefinose Gravity Column, were enzyme digested at room temperature overnight. All purified protein were dissolved in sterile phosphate-buffered saline (PBS), were measured their concentration and stored at -80°C.
Localized surface plasmon resonance (LSPR) Assays48
To examine the direct interaction between STAT3 and SNX3-retromer, LSPR assays were conducted on an OpenSPR system (Nicoya Lifesciences, Waterloo, Canada). Recombinant proteins STAT3 (117-770aa, 576-678aa), SNX3 and VPS35 served as the ligand and were respectively immobilized on a gold nanoparticle sensor chip via capture-coupling. Subsequently, the recombinant protein SNX3, VPS35 and VPS26 at different concentrations were sequentially injected into the chamber in running buffer (filtered PBS) with a constant flow rate of 20 μL/min, and were passed over the sensor (about 5 min) for the association of two protein. Following each recombinant protein injection (all concentrations were performed in triplicate), the chip was completely dissociated with the complex and regenerated by injecting hydrochloric acid (pH 2.0). As recommended by the manufacturer, the results were analyzed by Trace Drawer software (Ridgeview Instruments AB). The kinetic parameters, including the association constant (ka), dissociation constant (kd) and affinity (KD, KD=kd/ka), were calculated by a simple 1: 1 diffusion corrected model, which adjusted to the wavelength shifts consisting with the varied concentration of protein.
Primary culture of neonatal rat cardiomyocytes (NRCMs)
As reported before30, NRCMs were isolated from the hearts of one to three-day-old SD rats. Cardiomyocytes were plated into 6-well microplates (Corning, USA) comprising Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% newborn calf serum (NBCS) and 5-bromodeoxyuridine (Sigma, #B5002, 0.1 mmol/L), at a density of 1×106 cells/well.
Plasmid Transfection and virus infections30
NRCMs were transfected with sh-SNX3 plasmid together with lipofectamine 3000 reagent (Invitrogen, USA) in OptiMEM medium as per the manufacturer’s instructions. The medium was changed to DMEM complete medium after eight to seventy two hours of transfection. NRCMs were infected with recombinant adenoviruses (including Flag-tagged SNX3, Flag-tagged sh-SNX3 or HA-tagged STAT3) at a multiplicity of infection (MOI) of 20 particles per 5 cells. Western blot and/or Quantitative RT-PCR were performed to confirm the efficiency of overexpression or depletion.
Mitochondrial respiration assay by Seahorse XF9649
The prepared cardiomyocytes were seeded in a Seahorse XF96 polystyrene cell culture microplate (Seahorse Bioscience, North Billerica, USA), and incubated in a cell culture incubator at 37°C overnight. The next day, the medium was changed to XF base Medium (Seahorse Bioscience) containing 10 mmol/L glucose (Sigma), 1 mmol/L pyruvate (Sigma), and 2 mmol/L glutamine (Sigma) for the detection of basal respiration. Some reagents were serially injected to detect ATP production (mitochondria respiration), maximal respiration and non-mitochondrial respiration, including oligomycin (2 μmol/L, inhibits ATP synthase (complex Ⅴ), FCCP (1 μmol/L, uncouples oxygen consumption from ATP production), and a mix of rotenone (0.5 μmol/L, inhibits complexes Ⅰ) and antimycin A (0.5 μmol/L, inhibits complexes Ⅲ). Proton leak and spare respiratory capacity were calculated by these parameters using a high-throughput XFe96 extracellular flux analyzer (Seahorse Bioscience).
Measurement of the cell surface area30,50,51
NRCMs were seeded in 24-well microplates, fixed with paraformaldehyde (4%, Beyotime, #P0099) for 10 min at room temperature. After permeabilizing with Triton X-100 (0.3%, Beyotime, #P0096) and blocking with goat serum (Beyotime, #C0265), the cells were incubated with primary antibody myosin light chain 2 (MLC2, Proteintech, #10906-1-AP) overnight at 4℃, treated with secondary antibody anti-alexa fluor 488 (Proteintech, #SA00013-6, for 2h) and rhodamine-phalloidin (0.1%, Invitrogen #R415, for 30 min) at room temperature. After washing with filtered PBS, NRCMs were mounted by using DAPI (CST, #4083). The images were taken using the High Content Screening System (Thermo Fisher Scientific, USA), and the cell surface area from randomly selected fields (fifty for each group) was analyzed by the built-in image analysis software.
Isolation of early endosomes by continuous density gradient centrifugation52,53
The nuclear and cytoplasmic protein was successively extracted from NRCMs using a kit (SC-003, Inventbiotech, MN, USA). The postnuclear fraction was suspended in buffer, and was used to a continuous sucrose density gradient. After centrifugation (210,000 g for 3 h at 4°C), a milky band should be visible at each interface. 24 consecutive fractions were collected from each interface into tubes, and were subjected to western blot analysis for detection of protein EEA1, which is an early endosomal marker.
Low temperature SDS-PAGE, Western blot and co-immunoprecipitation (co-IP) analysis
Low-temperature SDS-PAGE was conducted to investigate the homodimers (STAT3/STAT3) or heterodimer (STAT3/STAT1). In short, total protein from NRCMs were incubated in loading buffer (without 2-mercaptoethanol) for 5 min at 37°C, were separated by 8% SDS-PAGE for 4-5 h at a constant current of 40 mA, and were transferred to a PVDF membranes (Millipore). The whole process of the experiment must be made under the low temperature. After blocking with 10% blocking buffer (Beyotime, #P0023B), the membranes were incubated with individual antibodies at 4°C overnight54.
Western blot analysis was performed as previously reported30,50. Immunoblots were labelled with the following primary antibodies: primary antibodies against p-JAK2 (Y1007 and Y1008, rabbit, diluted 1:500, #3776), JAK2 (rabbit, diluted 1:1,000, #3230), gp130 (rabbit, diluted 1:1,000, #3732), p-STAT3 (Y705, rabbit, diluted 1:1,000, #9145), STAT3 (rabbit, diluted 1:2,000, #9139) were bought form CST. Primary antibodies against clathrin heavy chain (CHC, rabbit, diluted 1:1,000), EEA1 (rabbit, diluted 1:1,000) and Rab5 (rabbit, diluted 1:1,000) were purchased from CST (endosomal marker antibody sampler kit, #12666). SNX3 (rabbit, diluted 1:1,000, #10772-1-AP), VPS26 (rabbit, diluted 1:800, #15915-1-AP) and VPS35 (rabbit, diluted 1:800, #10236-1-AP) were products of Proteintech. Primary antibodies against importin α3 (rabbit, diluted 1:1,000, #I9783), HA (rabbit, diluted 1:5,000, #H6908) and α-tubulin (mouse, diluted 1:5,000, #T8203) were purchased from Sigma-Aldrich. Anti-Flag (mouse, diluted 1:5,000, #PM185), anti-Lamin B1 (rabbit, diluted 1:1,000, # PM064) were purchased from MBL. The enzyme horseradish peroxidase (HRP) -conjugated secondary antibodies (CST, #7074 and #7076) were applied to chemiluminescence detection and the protein band intensities were quantified by LabWorks software (Bio-Rad, USA).
For co-IP, anti-EEA1 (rabbit, diluted 1:50), anti-CHC (rabbit, diluted 1:50), anti-Rab5 (rabbit, diluted 1:20) were purchased from CST (endosomal marker antibody sampler kit, #12666) Anti-VPS26 (rabbit, diluted 1:20, #15915-1-AP) and anti-VPS35 (rabbit, diluted 1:20, #10236-1-AP) were products of Proteintech. Anti-Flag (rabbit, diluted 1:50, #PM185) was purchased from MBL. Anti-HA (rabbit, diluted 1:50, #H6908) was purchased from Sigma-Aldrich. The rabbit normal IgG (#3900) and mouse normal IgG (#53484) were purchased from CST. NRCMs were harvested with IP lysis buffer (Beyotime, # P0013) supplemented with protease and phosphatase inhibitor cocktails (Bimake, #B14012 and #B15002). After clarification by centrifugation, 400-600 μg of total protein cell lysate were incubated with the indicated primary antibodies overnight at 4°C, and were incubated with protein G-agarose beads (Pierce, Rockford, IL, USA) at 4°C for 4 h. Normal IgG was served as an control. The immunoprecipitated proteins were detected by western blot or mass spectrum analysis.
Immunofluorescence (IF) assay
NRCMs were cultured in chamber slides (ThermoFisher Scientific). After treatment, cells were washed with filtered PBS for 3 times, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.3% Triton X-100 for 5 min and followed by blocking with goat serum for 1 h at room temperature. The cells were further treated with the following primary antibodies overnight at 4℃: STAT3 (CST, #9139), SNX3 (Proteintech, #10772-1-AP), VPS35 (Proteintech, #10236-1-AP), VPS26 (Proteintech, #15915-1-AP), EEA1 (CST, #12666), Rab5 (CST, #12666), Rab7 (CST, #12666), CHC (CST, #12666), Lamp-2 (Proteintech, #66301-1-Ig), gp130 (CST, #3732), JAK2 (CST, #3230) and importin α3 (Sigma-Aldrich, #I9783). Fluorescence emitted by fluorescence-conjugated secondary antibodies (Proteintech, #SA00013-6 and #SA00013-8) at room temperature for 2 h. The slides were mounted with DAPI (CST, #4083) and were observed by a confocal microscope (Zeiss, Germany) or EVOS FL Auto (Life Technologies).
Total RNA isolation, cDNA Synthesis and real-time polymerase chain reaction (qPCR)
Total RNA was extracted from snap-frozen cardiac tissues or NRCMs by using Trizol reagent (Invitrogen, #15596026), and its concentration was measured with a Nanodrop 2000 (Thermo Fisher Scientific, USA). The RNA extract (1000 ng) was reversely transcribed to first strand cDNA using the One-step Reverse Transcription (RT) Kit (Thermo Fisher Scientific, USA). Quantitative SYBR Green-based PCR (TOYOBO, Janpan) was conducted on the iCycler iQ system (Bio-Rad, USA). GAPDH was used as reference gene. All PCR assays were performed in triplicate. Data were analyzed using the 2-ΔΔCT method. The oligonucleotide sequences are synthesized by Sangon (Shanghai, China), and listed in table S7.
Dual-luciferase reporter gene assay
The conserved DNA binding sequence of rats STAT3 (TTCCGGGAA) were subcloned into pGL3 Basic plasmid (Promega, #E1751)55. NRCMs were seeded at 5×104 cells per well into 96-well microplates, and were transiently co-transfected with the luciferase reporter (100 ng/well) and pRL-TK reporter constructs (Promega, E2241) at 20 ng/well. The total content of transfected DNA was normalized by empty vector. After the indicated treatments, the luciferase activity was determined by the dual-luciferase reporter assay system (Promega, #E1980) on a microplate reader (TECAN Infinite M1000).
Statistical Analysis
Graph Pad Prism 6.0 (Graph Pad software) or SPSS Version 21 was used for statistical analysis. Normality of the obtained data was assessed using a Shapiro-Wilk test. When normality was confirmed, statistical differences among groups were analyzed using either Student's t test (for two groups) or one (or two) -way analysis of variance (ANOVA, for more than two groups). Otherwise, the non-parametric test Kruskal-Wallis test followed by the Dunn’s post-hot test was used to correct for multiple comparisons. In all cases, differences were considered statistically significant at a P value less than 0.05.