CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway

doi:10.21203/rs.3.rs-2004988/v1

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CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway

https://doi.org/10.21203/rs.3.rs-2004988/v1

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Pathological myocardial hypertrophy is a common cardiovascular disease that can progress to heart failure. At present, there is no ideal therapeutic drug in clinical practice. This study aimed to find new therapeutic targets for pathological myocardial hypertrophy. CD9 is a member of the tetraspanin protein family which is widely studied in inflammation and cancer, but has not been studied in pathological cardiac hypertrophy. In this study, we found that the expression of CD9 increased in TAC myocardial tissue. Knockdown of CD9 can alleviate the damage of cardiac function in TAC model, and can reduce heart weight, cardiomyocyte size and degree of fibrosis; overexpression of CD9 can aggravate the damage of cardiac function in TAC model, and can increase cardiac weight, cardiomyocyte size and degree of fibrosis. Mechanistically, Co-IP results showed that CD9 and GP130 can bind to each other in cardiomyocytes, knockdown of CD9 can reduce the protein level of GP130 and phosphorylation of STAT3, and overexpression of CD9 can increase the protein level of GP130 and phosphorylation of STAT3 in vivo and in vitro. Knockdown of GP130 reversed the aggravating effect of CD9 on pathological cardiac hypertrophy. Therefore, we conclude that CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway and it may serve as a therapeutic target for pathological cardiac hypertrophy.

CD9

Pathological cardiac hypertrophy

TAC

GP130

STAT3

The increase of cardiomyocyte size in response to an increased workload called cardiac hypertrophy. Hypertrophy initially develops as an adaptive response to physiological and pathological stimuli, but pathological cardiac hypertrophy, which is associated with increased interstitial fibrosis, cell death and cardiac dysfunction, is a key risk factor for heart failure[1]. Due to the poor overall prognosis of heart failure, it is significant to identify new therapeutic targets to prevent or reverse cardiac hypertrophy and heart failure.

The pathogenesis of pathological cardiac hypertrophy is complex, and the whole process involves abnormal protein synthesis, participation of various cytokines, excessive activation of signal transduction pathways, re-expression of cardiac embryonic bases etc. There are many signaling cascades/proteins implicated in mediating pathological cardiac hypertrophy. Among them, GP130/STAT3 signaling plays a key role in pathological cardiac hypertrophy[2, 3]. In addition to its cytoprotective action, GP130-dependent signaling induces cardiomyocyte hypertrophy[4]. Cecile Ancey et al. demonstrated that GP130 activation in human cardiac cells leads to cardiomyocyte hypertrophy[5]. GP130 receptor mediated signaling promotes cardiomyocyte survival, induces hypertrophy, modulates cardiac extracellular matrix and cardiac function[6]. GP130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway[7].

CD9 is a transmembrane glycoprotein belonging to the tetraspanin family[8]. Depending on the cell type and associated molecules, CD9 has a wide variety of biological activities such as cell adhesion, cell migration, cellular signaling, inflammation and tumor related processes[9–12]. Yu Shi et al. found that CD9 stabilizes GP130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells[13]. Jae-Ryong Kim et al. found that CD9 expression may play an important role in vascular senescence and pathogenesis of atherosclerosis[14]. Jung Hee Cho et al. found that CD9 induces cellular senescence and aggravates atherosclerotic plaque formation[15].

So, we speculate that CD9 may serve as a therapeutic target for pathological cardiac hypertrophy. In this study we want to explore whether CD9 play a role in pathological myocardial hypertrophy.

2.1 Animals

Eight-week-old C57BL/6J mice provided by HFK Bioscience (Beijing, China) were housed in a specified pathogen-free (SPF) animal room with a 12-hour light/12-hour dark cycle and free access to food and water. SD rats within 1 to 2 days after birth were used for the isolation of primary myocardial cells. All experimental procedures in this study were approved by the Animal Use Subcommittee of Tongji Medical College of Huazhong University of Science and Technology.

2.2 Recombinant adenovirus and adeno-associated virus production

Recombinant adenovirus containing CD9, CD9 shRNA (a short hairpin RNA targeting CD9), green fluorescent protein (GFP, as a negative control) or scramble shRNA (as a nonspecific control) was applied to infect primary cardiomyocyte in vitro.

The recombinant adeno-associated virus serotype 9 (rAAV9, Obio Technology) was used to achieve the overexpression and knockdown of CD9 in vivo. The recombinant adeno-associated virus (AAV) system (type 9), which contained control and CD9 with a heart-specific promoter (TNT promoter) or scramble shRNA and CD9 shRNA, was employed to manipulate the expression of CD9 in vivo via tail vein injection. Mice were injected via the tail vein with 100 µl of virus containing 2×10¹¹ VG of the AAV9 vector genomes.

The recombinant adeno-associated virus (AAV) system (type 9), which contained scramble shRNA and GP130 shRNA, was employed to manipulate the expression of GP130 in vivo via tail vein injection. Mice were injected via the tail vein with 100 µl of virus containing 2×10¹¹ VG of the AAV9 vector genomes. All adenoviruses and adeno-associated viruses were provided by Obio Technology Corp, Ltd. (Shanghai, China).

2.3 Transaortic constriction (TAC) model

C57BL/6J mice aged 8–10 weeks were randomly divided into four groups. 0.5% pentobarbital was injected into the abdominal cavity for anesthesia and fixed on the surgical plate, and the anterior area was depilated. The skin was cut on the upper limbs of the mice to separate the soft tissues on both sides of the skin. A 7 − 0 medical suture was inserted into the adipose tissue of the upper margin of the aorta and out of the triangular area of the adipose soft tissue of the lower margin of the aorta with ophthalmic tweezers. The suture was pulled out and the original route was withdrawn from the threading device. The 27G needle was placed at the aortic arch and the 7 − 0 suture was tied with a surgical knot. After the two knots were tightened, the excess suture was cut and the 27G needle was withdrawn. 4 − 0 medical sutures were used to suture the left and right pectoralis major muscles of mice. The skin was sutured, disinfected with alcohol cotton balls, and the mice were placed on a heating pad at 37°C until they recovered from anesthesia. The heart function of the mice was detected by ultrasound 4 weeks after TAC and before surgery, and the body weight, heart weight and lung weight of the mice were weighed.

2.4 Echocardiography and electrocardiogram

After the mice were anesthetized with isoflurane, the hair in the anterior chest area was removed after the mice were in a stable state. And they were fixed to the ultrasound examination table in the supine position, and then the ultrasound coupling agent was applied to the anterior chest area, and the heart rate was maintained at about 400 bpm. The long-axis and short-axis B-mode ultrasound images of the mouse heart were collected by Vevo2100 small animal ultrasound, and the cardiac ejection fraction (EF%), fractional shortening (FS%), and left ventricular end-diastolic phase were measured and calculated. Left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) were used to analyze the cardiac function of mice.

2.5 Histological assessments

Tissue sections were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin by Bios Biological Company. Serial 5 mm-thick transverse sections were stained in batches with hematoxylin and eosin (H&E). Digital photographs were taken by an inverted microscope (OLYMPUS IX73). ImageJ software was used for morphometric analyses.

2.6 Sirius red staining

Tissue sections were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin by Bios Biological Company. Serial 5 mm-thick transverse sections were stained in batches with lapis lazuli blue dye for 5–10 min, washed with distilled water for 3 times, and stained in batches with Sirius red saturated picric acid dye for 15–30 min. And then anhydrous ethanol direct differentiation and dehydration, neutral gum sealing. Digital photographs were taken by an inverted microscope (OLYMPUS IX73). ImageJ software was used for morphometric analyses.

2.7 Isolation and culture of primary myocardial cells

Wipe the ultra-clean table with a sterile gauze soaked with 75% medical alcohol. Place the pre-autoclaving and sterilized and dried surgical instruments into the ultra-clean table for uv irradiation for 30 min. Preheat the pre-configured complete medium containing 10% FBS, PBS buffer and D-Hanks buffer in a 37°C water bath. Type II collagenase was dissolved with sterile D-Hanks solution, and then fully dissolved, filtered with a 0.22 µm aperture filter for sterilization and reserve, with the concentration of 1 g/L. Take an appropriate number of 6-well plates according to the requirements of the experiment, add 1ml of high-pressure 1% bacteria-free liquid to each well, and place it in a 37°C incubator for 2 h. SD rats within 1 to 2 days after birth were soaked in 75% alcohol for several seconds. The rats were quickly removed and their head and neck were fixed with the left thumb and food. The rats were cut open in the chest area with ophthalmology, and their hearts could be ejected from the cavity by gently pushing the back of the left middle finger. After the heart was removed, it was immediately put into a cell culture dish containing pre-cooled D-Hanks solution. Residual blood was cleaned, useless tissues around the heart were removed, and only the tip part was kept as far as possible. The clipped cardiac apical tissue was placed in a new cell culture dish, and 2 mL type II collagenase was added to the dish. The clipped cardiac tissue was placed in a 37°C water bath for pre-digestion for 2 min, and the digestive fluid was discarded after the heart tissue naturally precipitated. 4 mL of new type II collagenase was added to the heart tissue, and the tissue was suspended by blowing up and down. The tissue was placed in a 37°C water bath for digestion for 10 min, during which the digestive liquid in the centrifugal tube was gently shaken to make the heart tissue complete. The liquid in the tube was centrifuged at 1000 RPM for 5 min. Collect the upper layer of liquid and add to the previously prepared fetal bovine serum to stop digestion, gently invert and mix. Repeat digestion for 5 to 6 times until the heart tissue block is completely digested. The filtrate was centrifuged at room temperature at 1000 RPM for 5 min. After discarding supernatant, precipitation was obtained by fully and gently re-suspending precipitation with a complete medium containing 10% FBS, slowly blowing and mixing with a rubber head dropper, and inoculated in the cell culture plate at an appropriate density, and placed in a 37°C incubator with 5% CO₂ concentration. The cells were incubated for 1 h in a 37°C cell culture box with 5% CO₂ ventilation concentration.

After microscopic observation, the cell suspension that was not attached to the wall was sucked out, the cells were counted, and inoculated into the culture plate according to the density of 1×10⁶/well of the 6-well plate. After conventional culture, the myocardial status was observed under microscope after cell adherence for 24 h. Obvious myocardial cell pulsation was observed, about 60–100 times /min. Corresponding intervention and treatment of cells could be given after fluid change.

After the suspension of non-adherent cells was removed, the remaining adherent cells were fibroblasts, and corresponding intervention and treatment could be given to the cells 24 hours later.

Using DMSO, Ang II was prepared into 50 mM storage solution and stored in -80°C refrigerator. Cells were treated with 50 µM working solution concentration for 24 h or 48 h to construct pathological myocardial hypertrophy model.

The stable cells were infected with Adenovirus Control (Ad Control), Adenovirus-CD9 (Ad CD9) and Adenovirus Scramble-shRNA (Ad Scr-sh) and Adenovirus CD9-shRNA (Ad CD9-sh) respectively. After 24 h of infection, the cardiomyocytes were stimulated with Ang II (50 µM) for 24 h. The cells were collected for further experiments.

2.8 Quantitative real-time RT-PCR (qRT-PCR)

Total RNA was extracted from mouse cells and heart tissues using TRIzol (D9108A, TaKaRa Bio). Reverse transcription of isolated RNA into complementary DNA (cDNA) was performed using the RR037A PrimeScript™ RT reagent Kit (Perfect Real Time) (TaKaRa). SYBR Green (Vazyme) was used to quantify the amplification products on a PRISM 7900 Sequence Detector System (Applied Biosystems, Foster City). All genes were quantified using β-actin as an internal control. Primer sequences are available upon request.

2.9 Co-immunoprecipitation (Co-IP) and Western blotting

The cells were lysed in ice-cold IP buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 0.5 mM β-glycerophosphate disodium salt hydrate and 1 mM phenylmethylsulfonyl) containing complete protease inhibitor (no. 04693132001, Roche) and centrifuged at 12,000 g for 15 min. The cell lysate was collected and incubated with protein G Sepharose beads (no. 11719416001, Roche) with the indicated antibody overnight at 4°C and then washed in immunoprecipitation buffer. The immune complexes were collected and immunized with the indicated antibody.

Whole-cell lysates were isolated from tissues or cells at the indicated times using RIPA lysis buffer with PhosSTOP phosphatase inhibitor (Roche Diagnostics, Barcelona, Spain) and complete protease inhibitor cocktail (Roche). The BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) was applied to determine protein concentrations. Equal amounts of protein were subjected to PAGE; after that, proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After incubation in nonfat milk in TBST to block the membranes, the membranes were incubated with the following primary antibodies: anti-CD9 (Affinity, AF5139), anti-ANP (Proteintech, 27426-1-AP), anti-GAPDH (Proteintech, 60004-1-Ig), anti-GP130 (Affinity, AF6291), anti-STAT3 (Proteintech, 10253-2-AP), anti-p-STAT3 (Tyr705) (Cell Signaling Technology, #73533) at 4ºC overnight. Then, the samples were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Chemiluminescence signals were detected by the ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA).

2.10 Statistical analysis

Data represented the mean ± SD and were compared between or among groups by two-tailed Student's t-test. P < 0.05 was considered statistically significant.

3.1 The expression of CD9 increased in TAC myocardial tissue

To explore the correlation between CD9 and pathological myocardial hypertrophy, the expression of CD9 and ANP in cardiac tissue four weeks after TAC surgery were detected by western blot and RT-PCR. The results showed that compared with Sham group, the expression of CD9 and ANP increased in cardiac tissue after TAC both in the mRNA and protein level (Fig. 1A-B). In the in vitro experiments, we found that the content of CD9 in cardiomyocytes was significantly higher than that in fibroblasts (Fig. 1C). Then we used Ang II to stimulate NRCMs to establish pathological myocardium hypertrophic model at the cellular level. The results showed that the expression of CD9 and ANP was increased in cardiomyocytes stimulated by Ang II in the mRNA and protein level (Fig. 1D-E).

3.2 Knockdown of CD9 ameliorated TAC-induced pathological cardiac hypertrophy

In order to verify the effect of CD9 on pressure-stressed myocardial hypertrophy in vivo, we injected C57BL/6J mice with AAV-CD9-shRNA and AAV-Scramble-shRNA virus through the tail vein, and performed TAC surgery on the mice 2 weeks later. The cardiac function of the mice was detected by ultrasound, and the results showed that compared with the Sham group, the LVEDd and LVESd were increased, and the myocardial systolic function EF% and FS% were decreased at 4 weeks after TAC. Meanwhile, compared with the TAC + AAV-Scramble-shRNA group, the LVEDd and LVESd were decreased, and the myocardial systolic function EF% and FS% were increased in the TAC + AAV-CD9-shRNA group, although one of them was not statistically different (Fig. 2A-D), suggesting that CD9 knockdown can alleviate the cardiac function damage after TAC. The mice were sacrificed 4 weeks after surgery to detect relevant indicators to clarify the effect of CD9 knockdown on pressure-stressed cardiac hypertrophy. As with the ultrasound results, the HW/BW, LW/BW and HW/TL were increased significantly at 4 weeks after TAC, and the ratio were decreased after CD9 knockdown, indicating that knockdown of CD9 reduced the heart weight of mice (Fig. 2E-G).

In order to further verify the effect of knockdown of CD9 on the size of cardiomyocytes after TAC, HE staining was performed on the cardiac tissue sections of mice in each group. Report the cross-sectional area was increased significantly at 4 weeks after TAC, and was decreased after CD9 knockdown (Fig. 2H-I).

In order to detect the effect of knockdown of CD9 on the degree of myocardial fibrosis in mice after TAC, the mouse heart tissue was stained with Sirius red. The results showed that the degree of interstitial fibrosis increased significantly 4 weeks after TAC, and the degree of interstitial fibrosis was significantly decreased in CD9 knockdown mice (Fig. 2J-K). RT-PCR showed that the expressions of ANP, BNP, MYH7, and Collagen Iα, Collagen III, Ctgf were increased in cardiac tissue after surgery, and knockdown of CD9 could reduce the levels of ANP, BNP, MYH7, and Collagen Iα, Collagen III, Ctgf (Fig. 2L-M).

3.3 Overexpression of CD9 aggravates TAC-induced pathological cardiac hypertrophy

To further investigate the effects of CD9 in TAC-induced pathological cardiac hypertrophy. We injected C57BL/6J mice with AAV-CD9 and AAV-Control virus through the tail vein, and the results showed that compared with the TAC + AAV-Control group, the LVEDd and LVESd were increased, and the myocardial systolic function EF% and FS% were decreased in the TAC + AAV-CD9 group (Fig. 3A-D), suggesting that CD9 can exacerbate the cardiac function damage after TAC. And the HW/BW, LW/BW and HW/TL were increased after CD9 overexpression (Fig. 3E-G). HE staining was performed on the cardiac tissue sections of mice in each group. The cross-sectional area was increased after CD9 overexpression (Fig. 3H-I). Sirius red results showed that the degree of interstitial fibrosis was significantly increased in CD9 overexpression mice (Fig. 3J-K). RT-PCR showed that CD9 could increase the levels of ANP, BNP, MYH7, and Collagen Iα, Collagen III, Ctgf (Fig. 3L-M).

3.4 CD9 promotes cardiac hypertrophy by regulating GP130 to activate STAT3

The above gain and loss of function experiments prove that overexpression of CD9 can aggravate the severity of pressure-overload induced myocardial hypertrophy. In order to explore its mechanism, it has been reported that CD9 could activate STAT3 by stabilizing GP130, and STAT3 may participate in pathological myocardial hypertrophy. GP130/STAT3 inhibition alleviated TAC-induced cardiac remodeling. Therefore, we aimed to confirm whether CD9 functions through GP130 signaling in cardiac hypertrophy. The co-IP experiment showed that CD9 and GP130 bind to each other in cardiomyocyte (Fig. 4A-B). It was further detected that the expression of GP130 and the phosphorylation level of STAT3 in cardiomyocyte were decreased when CD9 was knocked down in cardiomyocytes under the stimulation of AngII (Fig. 4C). And overexpression of CD9 can increase the expression of GP130 and the phosphorylation levels of STAT3 (Fig. 4D). Correspondingly, GP130 levels and STAT3 phosphorylation levels were decreased when knockdown of CD9 in the TAC model (Fig. 4E), and GP130 levels and STAT3 phosphorylation levels increased when overexpression of CD9 in the TAC model (Fig. 4F).

3.5 Knockdown of GP130 reverses CD9-induced pathological cardiac hypertrophy

The above results indicate that CD9 may be involved in pathological cardiac hypertrophy through GP130/STAT3 signaling. To further confirm it, we knocked down GP130 on the basis of overexpression of CD9 in TAC model. The results showed that knockdown of GP130 can reverse the increase of LVEDd and LVESd caused by overexpression of CD9, and the increase of myocardial contractile function EF% and FS% (Fig. 5A-D). The increased HW/BW, LW/BW and HW/TL by overexpressing CD9 were also reversed by knockdown of GP130 (Fig. 5E-G). Correspondingly, the increased cardiomyocyte cross-sectional area and cardiac fibrosis in CD9-overexpressing mice was also reversed by knockdown of GP130 (Fig. 5H-K). At the same time, and the elevated levels of ANP, BNP, MYH7, Collagen Iα, Collagen III and CTGF in CD9-overexpressing mice were reversed by knockdown of GP130 (Fig. 5L-M). It was further detected that the phosphorylation level of STAT3 in CD9-overexpressing mice were reversed by knockdown of GP130 (Fig. 5N). So the effect of CD9 on the aggravation of pathological cardiac hypertrophy was reversed by knockdown of GP130.

In this study, our result showed that the expression of CD9 increased in TAC myocardial tissue. In addition, knockdown CD9 ameliorated TAC-induced pathological cardiac hypertrophy, while overexpression of CD9 aggravates TAC-induced pathological cardiac hypertrophy. Mechanistically, our results showed CD9 promotes cardiac hypertrophy partially by regulating GP130 to activate STAT3.

When the cardiac pressure load increases, in order to ensure the cardiac ejection, the early myocardial cell hypertrophy is called compensatory myocardial hypertrophy. The number of organelles in hypertrophic cells increases and their functions are enhanced. However, the functional compensatory effect of cell hypertrophy is also limited. As the pressure load continues, when the cardiomyocytes are excessively hypertrophic, the blood supply to the cardiomyocytes is relatively deficient, which eventually leads to the overloading of the myocardium and the reduction of contractility. When the normal ejection fraction cannot be met, insufficiency is induced, that is, decompensated pathological myocardial hypertrophy. Pathological myocardial hypertrophy continues to progress, leading to heart failure[1].

The pathophysiological relevance of GP130 signaling pathways in the development of heart failure was clearly demonstrated by means of different genetic approaches. Such as Hiroki Uozumi et al. showed that transgenic mice, which express a dominant negative mutant of gp130 in the heart appeared a protect effect against pressure overload-induced hypertrophy, and suggested that GP130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway[7], Our results showed CD9 promotes cardiac hypertrophy partially by regulating GP130 to activate STAT3, which was consistent with Hiroki Uozumi et al.’s results. Opposite to GP130 deficiency, continuous activation of GP130 in the heart, achieved by combined overexpression of both IL-6 and the IL-6 receptor, leads to ventricular hypertrophy[16]. In addition, Keita Kunisada et al. showed that cardiac-specific transgenic mice over-expressing STAT3 displayed cardiac hypertrophy that was protective against doxorubicin-induced cardiomyopathy[17]. These experimental data clearly demonstrate that GP130 receptor signaling is required for compensatory hypertrophy and cardioprotection during episodes of pressure overload. However, other data in the literature suggests that the GP130/JAK/STAT pathway has a protective role in the heart [6, 18, 19]. Hence, the role of the GP130/STAT3 pathway in the pressure overload-induced hypertrophy remains controversial. Recently, Shengqi Huo et al. showed that IL-6/gp130/STAT3 inhibition by raloxifene alleviated TAC-induced myocardial inflammation, cardiac remodeling, and dysfunction[20]. We speculate that the GP130/STAT3 pathway is protective in the compensatory phase of hypertrophy, but had side effect in pathological cardiac hypertrophy and even heart failure.

Binding of the ligand to the GP130 and LIF receptor complex triggers dimerization and leads to activation of Janus kinase (JAK). Activated JAKs, in turn, rapidly phosphorylate the tyrosine residues of these receptors, which subsequently recruit various signaling molecules, including signal transducer and activator of transcription 3 (STAT3), to the receptor complex. Activated STAT3 dimers translocate to the nucleus, where they lead to transcriptional activation of downstream target genes[21]. JAK-mediated activation of the GP130 signaling pathway in pressure overload has been well described, and cessation of activation of this pathway to prevent GP130 cytokine overstimulation may have an independent pathological effect on cardiac function[4]. Our study showed CD9 may serve as a therapeutic target for pathological cardiac hypertrophy.

In summary, the present study has demonstrated for the first time, to our knowledge, that CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway. These findings, therefore, identify CD9 as a potentially important therapeutic target for treating pathological cardiac hypertrophy. CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway.

Funding

This study was funded by the National Natural Science Foundation of China (No: 82000050, No: 82170239 and No: 81900257).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

C. Zhang and Z. Liu conceived and designed the research, Y. Li, K. Huang and L. Kong performed the experiments, Y. Li, K. Huang, L. Kong, Z. Hao, Y. Zhou, J. Shangguan, L. Gao, M. Wang, Y. Kang and X. Li contributed to analysis the data, C. Zhang and Z. Liu wrote and revised the manuscript. All the authors gave final approval.

Data Availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. All experimental procedures in this study were approved by the Animal Use Subcommittee of Tongji Medical College of Huazhong University of Science and Technology.

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CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signalling pathway

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Recombinant adenovirus and adeno-associated virus production

2.3 Transaortic constriction (TAC) model

2.4 Echocardiography and electrocardiogram

2.5 Histological assessments

2.6 Sirius red staining

2.7 Isolation and culture of primary myocardial cells

2.8 Quantitative real-time RT-PCR (qRT-PCR)

2.9 Co-immunoprecipitation (Co-IP) and Western blotting

2.10 Statistical analysis

3 Results

3.1 The expression of CD9 increased in TAC myocardial tissue

3.2 Knockdown of CD9 ameliorated TAC-induced pathological cardiac hypertrophy

3.3 Overexpression of CD9 aggravates TAC-induced pathological cardiac hypertrophy

3.4 CD9 promotes cardiac hypertrophy by regulating GP130 to activate STAT3

3.5 Knockdown of GP130 reverses CD9-induced pathological cardiac hypertrophy

Discussion

Declarations

References

Supplementary Files

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