The interaction protein of SORBS2 in myocardial tissue to find out the pathogenic mechanism of LVNC disease

Mass spectrometry identification experiment

After silver staining and cutting adhesive, each adhesive strip was enzymatically decomposed. The IP products were detected by mass spectrometry on the computer, and then combined to search the database. The strip was destained and dried at room temperature (RT). Reduction/alkylation was carried out, and NH₄HCO₃ was added, followed by shaking for 5 min. The solution was discarded and strips dried at RT. Trypsin solution was added and incubation was at 4° C for 60 min. Overnight enzymatic hydrolysis was at 37° C. Extract enzyme-hydrolyzed polypeptide. Centrifugal concentration drying. Sample loading processing. After vacuum drying, 0.1% FA was added to redissolve the sample, and 1-2 μg of sample were put on the instrument. The separation was performed with EASY nLC 1200 (Thermo Scientific, USA) using a self-filled Trap column (C18, 5 μm, 100 μm×2 cm) and an analysis column (C18, 1.9 um, 75 μm×20 cm) at a flow rate of 200 NL/min. The mass spectrometer was Orbitrap Fusion Lumos (Thermo Scientific, USA). The Data Dependent Acquisition (DDA) mode was adopted for the detection of tandem mass spectrometry. Full sweep resolution was 60,000 (FWHM), and the mass/charge ratio range was set at M/Z 375-1600, and collision energy was set at 30% in HCD fragmentation mode.

Screening for proteins that interact with SORBS2

The myocardial tissue of normal adult mice at 12 weeks of age was analyzed by mass spectrometry. GO (Biological Process, Molecular Function, Cellular Component) analysis and Pathway analysis were performed for the specifically detected proteins, and bioinformation annotation and analysis were performed at two levels. In this way, more important proteins were selected for further study. The interacting proteins were screened by mass spectrometry.

Western blotting

The cells were harvested and washed twice with PBS. They were then lysed with cold RIPA and centrifuged at 12,000 g at 4° C for 10 min. The supernatants were collected and assayed for protein by the BCA method. The tissue and cell homogenates were separated by SDS-PAGE (4-12% gradient) and transferred onto a PVDF membrane. The PVDF membranes were blocked by TBS-T with 5% milk for about 2 h. The PVDF membranes were then incubated with antibodies overnight at 4° C and incubated with secondary HRP-IgG antibodies (CST Biotechnolog, USA) for 2 h at RT.

The primary antibodies were as follows: SORBS2 (1:1000; Proteintech); YWHAQ (1:1000; Proteintech); GAPDH (1:1000; Proteintech); β-tubulin (1:1000; CST).

Immunofluorescence

Cells were cultured on glass coverslips with gelatin, fixed for 15 min in cold 4% paraformaldehyde, washed with PBS for 10 min, incubated with 5% normal goat serum (NGS; ZSGB-BIO, Beijing, China) in PBS for 1 h at RT, and then washed with PBS for 10 min. Cells were then incubated with the primary antibody overnight at 4° C, washed with PBS for 10 min, incubated with IgG peroxidase-conjugated secondary antibody for 1 h, and washed with PBS for 10 min. The cells were examined with a confocal scanning laser microscope (Sp8; Leica, Germany). The immunohistochemistry procedure was similar to that described above. The myocardial samples were fixed in 10% neutral buffered formalin. They were paraffin embedded after using alcohol and xylene gradients for dehydration. They were then blocked with 0.05% H2O2.

Immunostaining was done with the following antibodies: SORBS2 (1:100; Proteintech) and sarcomeric α-actinin (1:100, Abcam), CLTC polyclonal antibody (1:100; Proteintech); β-tubulin (1:100;CST); YWHAQ (1:200;CST); Oct4A (1:100;CST); MLC-2V (1:100; Proteintech); CTNI (1:100; Proteintech); CTNT (1:100; Proteintech); Sox2 (1:100; CST); SSEA (1:100; CST); Alexa 488-conjugated goat anti-mouse (Yeasen, Shanghai, China); Alexa 488-conjugated goat anti-rabbit (Yeasen, Shanghai); Alexa 594-conjugated goat anti-mouse (Yeasen, Shanghai); and Alexa 594-conjugated goat anti-rabbit (Yeasen, Shanghai). The cell nuclei were counterstained with 0.1% 4’,6-diamidino-2-phenylindole (DAPI; Yeasen, Shanghai).

Cell culture

The human embryonic stem cells were cultured in TeSR™-E8™ Basal Medium supplemented with TeSR™-E8™ media (StemCell Technologies, Cat. # 05990). Once the stem cells reached 70% confluency, they were then prepared to be induced to differentiate into hESC-CMs. RPMI1640 containing 3 μM CHIR-99021HCl (Selleck Chem, Cat. # S2924), and 2% B-27 minus insulin (Gibco, USA) as the culture medium was used to induce hESC. After 3 days, the cells were cultured in RPMI1640 containing 5 μM IWR-1-endo (Selleck Chem, S7086) and 2% B-27 minus insulin (Gibco). Two days later, the medium was replaced with RPMI1640 containing 2% B-27 minus insulin (Gibco). From then on, the medium was changed every 3 days with RPMI1640 plus 1× B-27 supplement. On day 16, collagenase type I (Sigma) was used to purify the cells, and the cells were then cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 15% fetal bovine serum (Gibco) at 37° C and 5% CO₂ in a humidified incubator, with the medium changed every day.

Cell transfection

To demonstrate the capacity of SORBS2 in cardiomyocytes, we created two appropriate carriers HBLV-GFP-PURO-NC and HBLV-h-3*flag-GFP-PURO to disrupt the cardiomyocytes. We purchased the lentivirus from HANBIO and strictly followed the instructions. The lentivirus could transfect after the hESC reached nearly 70% confluence with 1×10⁶ viruses per well of a 6-well plate. At 48 h after transfection, green fluorescence intensity could be observed under a fluorescence microscope. The transfected target cells were then isolated to proceed with the next experiments.

Co-immunoprecipitation

Co-IP assays were carried out using the 88804 Pierce Classic Magnetic IP/Co-IP Kit, Thermo [14]. The antibodies used were as follows: IP: rabbit polyclonal anti-SORBS2 (2 μg; Proteintech); rabbit polyclonal anti-YWHAQ (2 μg; Proteintech); IB: rabbit polyclonal anti-SORBS2 (1:1000; Proteintech); and rabbit monoclonal anti-YWHAQ (1:1000; CST). Heart tissues were lysed in ice-cold lysis buffer. Protein was collected by centrifugation at 12,000 rpm for 10 min at 4° C. The lysates were then incubated with 5 μg of antibodies pre-bound to protein A/G-Sepharose beads overnight at 4%. Beads were washed four times with ice-cold washing buffer, and eluted in SDS-PAGE sample repeat (50 mM Tris–HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 10 mM dithiothreitol). The precipitates were subjected to immunoblotting with antibodies.

Statistical analysis

All data were analyzed with SPSS Statistics 22 software. One-way analysis of variance (ANOVA) was used to evaluate the differences between the two groups. In all the analyses, P-value<0.05 and P-value<0.01 were considered indicative of a statistically significant difference.

Screening for proteins interacting with SORBS2 by mass spectrometry

In this study, we performed mass spectrometric identification of SORBS2-interacting proteins in normal mouse cardiac muscle tissue and hESC-CMs. For the specifically detected proteins, GO (Biological Process, Molecular Function, Cellular Component) analysis and pathway analysis were performed, and biological information annotation was conducted at two levels (Figure 1A, 1B). Thus, more important proteins were singled out for further study. Therefore, we used the myocardial tissue to identify the interacting proteins by mass spectrometry (Figure 1C).

Figure 1. Proteins interacting with SORBS2 were screened by IP experiment. (A) IP experiment screened the signal pathways that were different from SORBS2. (B) The Venn diagram shows the difference between the two groups of IgG and IP. (C) Proteins interacting with SORBS2 were screened by bioinformatics database. The main enrichment pathway of the proteins interacting with SORBS2 was screened by IP experiments.

These data suggested that as a specific protein up-regulated in LVNC heart tissue, SORBS2 may be associated with this disease. The proteins that are highly correlated with SORBS2 may play a certain role in the occurrence of diseases.

Proteins that interact with SORBS2

Some proteins interacting with SORBS2 were found by preliminary screening, and co-IP was further used to verify the interaction between them and SORBS2. For several proteins we focused on, the immunofluorescence technique was used for detection in hESC-CMs to re-confirm that the related proteins interacted with SORBS2. The proteins interacting with SORBS2 were screened by bioinformatics and online websites. In addition, 6 proteins highly correlated with SORBS2 were screened by IP: MYH7, YWHAQ, DES, ACTN2, TUBB2A and CLTC (Figure 2A).

Figure 2. Proteins that interact with SORBS2. (A) The proteins interacting with SORBS2 were screened according to the evaluation score and reliability analysis. We focused on YWHAQ interacting with SORBS2.

According to previous reports, TUBB2A does interact with SORBS2 [9]; moreover, 14-3-3 family knockout mice can have the phenotype of LVNC disease [15]. SORBS2 interacts with YWHAB and YWHAZ. Both YWHAB and YWHAZ belong to the 14-3-3 family of proteins, of which YWHAE (14-3-3) has been reported to be associated with the LVNC phenotype. In E12.5 embryonic mice, 14-3-3 deletion showed incomplete densification. Therefore, we focused on the 14-3-3 family as a protein highly correlated with SORBS2 (Figure 2A). These data suggest that YWHAQ, as a protein that interacts with SORBS2, may be involved in the pathogenesis of related diseases.

Embryonic stem cells induced to differentiate into cardiomyocytes

Next, we used hESC-CMs as the carrier in in vitro experiments to study whether overexpressed SORBS2 would affect cell phenotype and function. The totipotency of human embryonic stem cells was demonstrated by immunofluorescence staining with four stem cell marker proteins: Oct4, Sox2, Nanog and SSEA (Figure 3A). This experiment indicated that hESC still retained stem cell totipotency and could be used in the following induction experiments.

Figure 3. Stem cells are induced to differentiate into cardiomyocytes. (A) Human embryonic stem cells (hESC) expressing Oct4, Sox2, Nanog and SSEA. (B) hESC-derived cardiomyocytes (hESC-CMs) expressing MLC-2v, α-actinin, cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT). Scale bars=25μm.

The induced cardiomyocytes showed high purity and continuous beating. Four myocardial cell protein markers, MLC-2V, α-actinin, cardiac troponin I (cTnI) and cardiac troponin T (cTnT), were detected by immunofluorescence staining. This indicated that the induction of differentiation into cardiomyocytes was successful and that the purity of the cardiomyocytes was relatively high (Figure 3B).

SORBS2 interacts with YWHAQ

We further determined the localization of the selected proteins and the expression location of SORBS2 in the induced cardiomyocytes using immunofluorescence staining. We found that β-tubulin, ACTN2, CLTC and YWHAQ showed co-localization with SORBS2 (Figure 4A). Meanwhile, we used the co-immunoprecipitation assay to verify the interaction between SORBS2 and YWHAQ. These data collectively demonstrated that SORBS2 interacted with YWHAQ in normal myocardium (Figure 4B).

Figure 4. SORBS2 interacts with YWHAQ. (A) To better understand β-tubulin, ACTN2, CLTL and YWHAQ, we next performed studies in hESC-derived cardiomyocytes (hESC-CMs). Immunofluorescence revealed a clear colocalization of SORBS2 with β-tubulin, ACTN2, CLTL and YWHAQ in the hESC-CMs. The SORBS2 protein (red) colocalized with β-tubulin (green), and the merged images are shown in yellow. (B) Extracted proteins from the cardiomyocytes ware untreated (input) or precipitated by an anti-Flag or anti-Myc antibody and then separated by 4-12% SDS-PAGE. Co-immunoprecipitation (Co-IP) of the heart tissue showing SORBS2 binding with YWHAQ.

The hESC-CMs were transfected with HBLV-h-SORBS2 lentivirus to induce overexpression of SORBS2 (Figure 5A). Figure 5B shows that the levels of SORBS2 were significantly increased in the hESC-CMs in which SORBS2 showed overexpression by immunofluorescence staining. The immunoblots showed an increased proportion of SORBS2 (Figure 5C). More dramatically, to better understand the relationship between SORBS2 and YWHAQ in hESC-CMs in which SORBS2 was overexpressed, we found decreased YWHAQ expression (Figure 6A, 6B). Our previous studies showed that as a specific up-regulated protein in LVNC heart tissue, SORBS2 may have a particular relationship to this disease. Moreover, as the protein YWHAQ has a strong SORBS2 correlation, it would undoubtedly provide a more favorable research basis in our future studies.

Figure 5. Expression of SORBS2 in hESC-CMs transfected with HBLV-h-SORBS2 was higher compared with the control groups. (A) Cardiomyocytes transfected with lentivirus. (B) Representative images of SORBS2 in hESC-CMs transfected with empty virus (control) and HBLV-h-SORBS2 (SORBS2-OE). (C) Western blotting detection of the expression levels of SORBS2 in overexpression group and control group. The protein level of SORBS2 was normalized to GAPDH for each sample. Statistical analysis of the expression of SORBS2 in hESC-CMs transfected with empty virus (control) and HBLV-h-SORBS2 (SORBS2-OE) (n =3 per group, * p < 0.05; Student's t-test). Data are shown as the mean ± SEM.

Figure 6. YWHAQ protein decreased in the group that overexpressed SORBS2. (A) The expression of YWHAQ inf immunoblots of hESC-derived cardiomyocytes (hESC-CMs). (B) Statistical analysis of the expression of YWHAQ in hESC-CMs transfected with HBLV-h-SORBS2 and control cells. (* *p < 0.01).