Inhibition of PHLPP1 ameliorates cardiac dysfunction via activation of the PI3K/Akt/mTOR signalling pathway in diabetic cardiomyopathy

Abstract

Background: Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a kind of serine/threonine phosphatase, whose dysregulation is accompanied with numerous human diseases. However, its role in diabetic cardiomyopathy remains unclear. We explored the underlying function and mechanism of PHLPP1 in diabetic cardiomyopathy (DCM).

Method: In vivo, Type 1 diabetic rats were induced by intraperitoneal injection of 60 mg/kg streptozotocin (STZ). Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down the expression of PHLPP1. In vitro, primary neonatal rat cardiomyocytes and H9C2 cells were incubated in 5.5 mmol/L glucose (normal glucose, NG) or 33.3 mmol/L glucose (high glucose, HG). PHLPP1 expression was inhibited by PHLPP1-siRNA to probe into the function of PHLPP1 in high glucose-induced apoptosis in H9c2 cells.

Results: Diabetic rats showed up-regulated PHLPP1 expression, left ventricular dysfunction, increased myocardial apoptosis and fibrosis. PHLPP1 inhibition alleviated cardiac dysfunction. Additionally, PHLPP1 inhibition significantly reduced HG-induced apoptosis and restored PI3K/AKT/mTOR pathway activity in H9c2 cells. Furthermore, pretreatment with LY294002, an inhibitor of PI3K/Akt/mTOR pathway, abolished the anti-apoptotic effect of PHLPP1 inhibition.

Conclusion: Our study indicated that PHLPP1 inhibition alleviated cardiac dysfunction via activating the PI3K/Akt/mTOR signalling pathway in DCM. Therefore, PHLPP1 may be a novel therapeutic target for human DCM.

Keywords: PHLPP1; PI3K/Akt/mTOR signal; apoptosis; diabetic cardiomyopathy; fibrosis.

Figure 1

PHLPP1 expression and pathology of control and diabetic rat hearts. A, Western blot analysis of PHLPP1 protein levels (n = 6 per group). B1, Heart size (scale bar: 3 mm, n = 8 per group). B2, HE staining of cross shaft of musculi papillares in heart (n = 8 per group). B3, Representative haematoxylin and eosin staining (HE) of longitudinal left ventricular (LV) sections (scale bar: 20 μm, n = 8 per group). B4, Representative HE staining of LV transverse sections (scale bar: 20 μm, n = 8 per group). C, Relative mRNA fold changes of β‐MHC (n = 6 per group). D, Relative mRNA fold changes of BNP (n = 6 per group). E, Quantitative analysis of cardiomyocyte cell diameter (n = 8 per group). Control: normal rats. DM: diabetes mellitus. shN.C: negative control shRNA. shPHLPP1: PHLPP1 shRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with controls; #P < .05 compared with DM or shN.C in DM

Figure 2

Echocardiographic measurements of control and diabetic rat hearts (n = 8 per group). A1, Representative 2D echocardiograms. A2, Representative M‐mode echocardiograms. A3, Representative pulse‐wave Doppler echocardiograms of mitral inflow. A4, Representative tissue Doppler echocardiograms. B, LV ejection fraction (LVEF). C, Fractional shortening (FS). D, Early‐to‐late mitral flow (E/A). E, Diastolic velocity ratio (E′/A′). F, Left ventricular end‐diastolic dimension (LVEDd). Control: normal rats. DM: diabetes mellitus. shN.C: negative control shRNA. shPHLPP1: PHLPP1 shRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with controls; #P < .05 compared with DM or shN.C in DM

Figure 3

Effects of PHLPP1 on myocardial apoptosis and fibrosis. A, Immunostaining of PHLPP1 (first row, n = 6) and cell apoptosis as determined by TUNEL assay (second‐fourth row, n = 5): apoptosis cell stained red; nuclei stained blue with DAPI. B, Cell apoptosis rate determined by TUNEL assay. C, The levels of cleaved caspase‐3 following PHLPP1 inhibition were measured by Western blot (n = 6). D, The levels of Bax and Bcl‐2 following PHLPP1 inhibition were measured by Western blot (n = 6). E, Representative Masson's trichrome staining (first row) and Sirius red staining (second and third rows) of the myocardium (n = 6). Immunostaining of collagen I (fourth row) and collagen III (fifth row) (n = 6). F, Quantification of Masson's trichrome staining (n = 6). G, Quantification of Sirius red staining (n = 6). H‐K, Western blot analysis of the protein expression of collagen I (F), collagen III (G), MMP2 (H) and MMP9 (I) (n = 6). Control: normal rats. DM: diabetes mellitus. shN.C: negative control shRNA. shPHLPP1: PHLPP1 shRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with control, and #P < .05 compared with DM or shN.C in DM

Figure 4

The effects of different glucose culture times on PHLPP1 expression. A, Western blot analysis of the expression of PHLPP1 in primary neonatal cardiomyocytes. B, Western blot analysis of the expression of PHLPP1 in H9c2 cells. C, Immunofluorescence of PHLPP1 in primary neonatal cardiomyocytes and H9c2 cells. PHLPP1 stained green in primary neonatal cardiomyocytes and red in H9c2 cells; nuclei stained blue with DAPI. All experiments were performed at least 3 times.NG: 5.5 mmol/L glucose, HG: 33.3 mmol/L glucose. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Dunnett's multiple‐to‐one comparison test. *P < .05 compared with NG

Figure 5

High glucose up‐regulates the expression of PHLPP1 via increasing ROS. A, The production of ROS. 6‐h high‐glucose treatment increased ROS production and this overproduction was inhibited under pretreatment with NAC. B, The protein expression levels of PHLPP1 when pretreated with NAC before 24 h HG‐stimuli. 24 High glucose increased PHLPP1 expression while pretreatment with NAC attenuated this overexpression. NG: 5.5 mmol/L glucose; HG: 33.3 mmol/L glucose; NAC: N‐acetyl cysteine. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with NG. #P < .05 compared with HG

Figure 6

TUNEL analyses and Western blotting demonstrated that PHLPP1 induced apoptosis in H9c2 cardiomyocytes. A‐B, The cell apoptosis rate detected by TUNEL were reduced 24 h after PHLPP1 inhibition. C, The expression of PHLPP1 detected by Western blotting. D‐E, The expression of cleaved caspase‐3 and the Bax/Bcl‐2 ratio was reduced after the expression of PHLPP1 was inhibited. All experiments were performed at least 3 times. NG: 5.5 mmol/L glucose; HG: 33.3 mmol/L glucose. siN.C: negative control siRNA. siPHLPP1: PHLPP1 siRNA. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with NG. #P < .05 compared with HG

Figure 7

Signal transduction mechanisms involved in the induction of apoptosis by PHLPP1. PHLPP1‐siRNA plasmids were transfected 24 h prior to HG stimulation in H9c2 cell. Western blot analyses of p‐PI3K, p‐Akt and p‐mTOR expression in H9c2 cardiomyoblasts. The expression of p‐PI3K, p‐Akt and p‐mTOR were increased after PHLPP1 inhibition. NG: 5.5 mmol/L glucose; HG: 33.3 mmol/L glucose. siN.C: negative control siRNA. siPHLPP1: PHLPP1 siRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with NG. #P < .05 compared with HG

Figure 8

TUNEL analyses and Western blotting demonstrated that LY294002 effectively reversed the anti‐apoptotic effect of PHLPP1 inhibition. A, The cell apoptosis rates detected by TUNEL were reduced after PHLPP1 inhibition; however, this reduction was reversed after pretreated with LY294002. B, The expression of cleaved caspase3 was reduced after PHLPP1 inhibition, while LY294002 reversed this reduction. C, The ratio of Bax to Bcl2 was reduced after PHLPP1 inhibition but it was enhanced when treatment with LY294002. NG: 5.5 mmol/L glucose; HG: 33.3 mmol/L glucose. siN.C: negative control siRNA. siPHLPP1: PHLPP1 siRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. *P < .05 compared with HG + siNG. #P < .05 compared with HG + siPHLPP1