TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway

Figures

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  Fig. 1. Inflammatory cell infiltration in the myocardium. A Representative images of inflammatory cell infiltration into the myocardium according to H&E staining from the different groups (n = 5 per group, magnification = 200×). The myocardial interstitium was mainly infiltrated by mononuclear macrophages. B Representative western blot image of Caspase-1 in the myocardium from the different groups. α-tubulin was used as a loading control. C Corresponding densitometric analysis of blots in B (n = 6 per group). The data are represented as the means ± SE; *P < 0.05

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  Fig. 2. Immunofluorescent double staining of TRPM2 and WGA in the myocardium. A Representative images of IF double staining of TRPM2 and WGA in mice hearts from the different groups. B Corresponding statistic analysis of TRPM2 fluorescence intensity in A (n = 5 per group). C Corresponding statistic analysis of cardiomyocyte size used WGA staining in A (n = 5 per group). The data are represented as the means ± SE; **P < 0.01 and ***P < 0.001

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  Fig. 3. The expression of TRPM2 and apoptosis-related proteins. A Representative western blot image of TRPM2 in the myocardium from the different groups. α-tubulin was used as a loading control. B Corresponding densitometric analysis of blots in B (n = 6 per group). C Representative western blot image of Bax, Bcl-2, and cleaved caspase-3 in the myocardium from the different groups. α-tubulin was used as a loading control. (F) Corresponding densitometric analysis of blots in C (n = 6 per group). The data are represented as the means ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001

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  Fig. 4. Myocardial apoptosis detected by the TUNEL staining. A Representative images of TUNEL staining in the myocardium from the different groups. α-actin staining was used for co-localization of cardiomyocytes and DAPI was used for nuclear staining. B Corresponding statistic analysis of cardiomyocyte apoptosis in A (n = 5 per group). The data are represented as the means ± SE; *P < 0.05 and **P < 0.01

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  Fig. 5. TRPM2 knockdown inhibited myocardial excessive autophagy in HFD/STZ-induced diabetic mice. A Immunohistochemical analysis of autophagy substrates P62 protein in mice hearts from the different groups (magnification = 200×). B Corresponding statistic analysis of P62 in A (n = 5 per group). C Representative western blot image of P62 in the myocardium in each group. α-tubulin was used as a loading control. D Heart homogenates were analyzed by western blot using an antibody against LC3 II/I and ULK1 proteins. α-tubulin was used as a loading control. E Corresponding densitometric analysis of blots in C (n = 6 per group). F, G Corresponding densitometric analysis of blots in D (n = 6 per group). The data are represented as the means ± SE; *P < 0.05 and **P < 0.01

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  Fig. 6. Cardiac function in mice measured by echocardiography. A Representative images by M-mode echocardiography in mice hearts from the different groups. B Representative images by pulsed wave Doppler echocardiography in mice hearts from the different groups. C–E Echocardiography analysis showing cardiac systolic dysfunction assessed by LVEF (C) and LVFS (D) diastolic dysfunction assessed by ratio of diastolic mitral annulus velocities (E′/A′) (E) (n = 5 per group). The data are represented as the means ± SE; *P < 0.05 and **P < 0.01

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  Fig. 7. Changes in MEK/ERK signaling pathway. A Representative western blot analyses of p-MEK1/2 (Ser217/221) and MEK1/2, p-ERK1/2 (Thr202/Tyr204) and ERK1/2 in mice heart homogenates from the different groups. GAPDH was used as a loading control. B Corresponding densitometric analysis of blots in A (n = 6 per group). The data are represented as the means ± SE; *P < 0.05 and **P < 0.01

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  Fig. 8. Changes in mTORC1 signaling pathway. A Representative western blot analyses of p-mTOR (Ser2448) and mTOR, Raptor, p-S6K1 (Thr389) and S6K1, and p-4EBP1 (Thr37/46) and 4EBP1 in mice heart homogenates from the different groups. β-actin was used as a loading control. B Corresponding densitometric analysis of blots in A (n = 6 per group). The data are represented as the means ± SE; *P < 0.05 and **P < 0.01

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  Fig. 9. High glucose-induced oxidative stress increased TRPM2 expression. Primary cardiomyocytes were treated with 45-mM high glucose (HG) for 72 h to simulate diabetic myocardial injury in vivo. In addition, 5-mmol/L N-acetylcysteine (NAC) was used to inhibit ROS produced by primary cardiomyocytes stimulated by HG, and 50-μmol/L H2O2 treatment was regarded as a positive control group inducing ROS production in primary cardiomyocytes. The medium was changed every 12 h to maintain H2O2 concentration. A, B Intracellular ROS production in primary cardiomyocytes was detected with DCF-DA reagent and corresponding statistic analysis. Compared to that in controls, ROS levels significantly increased after HG stimulation for 72 h in primary cardiomyocytes. There was no significant change in the high osmotic pressure group (mannitol treatment) compared to control group, but the ROS level in the HG group was decreased by pre-incubation with NAC. H2O2-positive control group could also increase the ROS level of cardiomyocytes, while NAC pre-incubation decreased ROS levels in H2O2-positive control group. C Representative western blots of P62 and LC3 protein in primary cardiomyocytes from the different…

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  Fig. 10. TRPM2 silencing defended against high glucose-induced apoptosis and autophagy inhibition in primary cardiomyocytes. Primary cardiomyocytes were infected with lentivirus for 72 h and then treated with normal glucose or 45-mM high glucose (HG) for 72 h. A, B The apoptosis in different treatment groups was detected by flow cytometry and corresponding statistic analysis. Compared to that in controls, the apoptosis level of primary cardiomyocytes was significantly increased after 45-mM HG stimulation for 72 h, and there was no significant change in the hyperosmotic pressure group. However, TRPM2 silencing by lentivirus infection of primary cardiomyocytes could resist the apoptosis of HG group. C The cell viability was measured by cell counting kit-8. Compared to that in controls, the cell viability of primary cardiomyocytes decreased significantly after stimulated with HG for 72 h, and there was no significant change in the hyperosmotic pressure group. However, the vitality of primary cardiomyocytes in HG group could be partially restored after TRPM2 silencing. D Representative western blots of TRPM2, Bcl-2, Bax, and cleaved Caspase-3 protein in primary cardiomyocytes from the…

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  Fig. 11. TRPM2 silencing attenuated high glucose-induced apoptosis and autophagy inhibition in cardiomyocytes via regulating the MEK/ERK pathway. A Representative blots of p-MEK1/2 (Ser217/221) and MEK1/2, p-ERK1/2 (Thr202/Tyr204), and ERK1/2 in high glucose (HG)-stimulated primary cardiomyocytes and corresponding densitometric analysis. α-tubulin was used as a loading control. Compared to that in controls, the protein expression levels of p-MEK1/2 and p-ERK1/2 were significantly increased after HG stimulation, which was reversed by TRPM2 silencing. B Representative blots of MEK/ERK signaling pathway in HG-stimulated cardiomyocytes pre-incubated with U0126 and corresponding densitometric analysis. α-tubulin was used as a loading control. U0126 was widely used as an inhibitor of MEK/ERK signaling pathway. NRVMs were treated with 20-µM U0126 for one hour to inhibit the MEK1/2 pathway before exposure to 45 mM of HG stimulation for 72 h. The protein expression levels of p-MEK1/2 and p-ERK1/2 were significantly decreased by U0126 treatment in HG-stimulated cardiomyocytes after HG stimulation. C The cell viability was measured by cell counting kit-8 and corresponding statistic analysis. D, E…

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  Fig. 12. TRPM2 silencing attenuated high glucose-induced apoptosis and autophagy inhibition in cardiomyocytes via regulating the mTORC1 pathway. A Representative blots of p-mTOR (Ser2448), Raptor, p-S6K1 (Thr389), and p-4EBP1 (Thr37/46) in high glucose (HG)-stimulated primary cardiomyocytes and corresponding densitometric analysis. β-actin was used as a loading control. Compared to that in controls, the protein expression levels of p-mTOR, Raptor, p-S6K1, and p-4EBP1 were significantly decreased after HG stimulation, which was restored by TRPM2 silencing. B Representative western blots of P62 and LC3 in HG-stimulated cardiomyocytes pre-incubated with MHY1485 and corresponding densitometric analysis. β-actin was used as a loading control. MHY1485 is widely used as an agonist of mTORC1 signaling pathway. NRVMs were treated with 20-µM MHY1485 for one hour to activate the mTORC1 pathway before exposure to 45 mM of HG stimulation for 72 h. Compared to that in controls, the protein expression level of P62 significantly increased; ratio of LC3-II/LC3-I was also significantly decreased, in primary cardiomyocytes after HG stimulation, which was partially reversed by MHY1485 treatment. C…