Roles of transient receptor potential channel 6 in glucose-induced cardiomyocyte injury

Abstract

Background: Diabetic cardiomyopathy (DCM) is a serious complication of end-stage diabetes that presents symptoms such as cardiac hypertrophy and heart failure. The transient receptor potential channel 6 (TRPC6) protein is a very important selective calcium channel that is closely related to the development of various cardiomyopathies.

Aim: To explore whether TRPC6 affects cardiomyocyte apoptosis and proliferation inhibition in DCM.

Methods: We compared cardiac function and myocardial pathological changes in wild-type mice and mice injected with streptozotocin (STZ), in addition to comparing the expression of TRPC6 and P-calmodulin-dependent protein kinase II (P-CaMKII) in them. At the same time, we treated H9C2 cardiomyocytes with high glucose and then evaluated the effects of addition of SAR, a TRPC6 inhibitor, and KN-93, a CaMKII inhibitor, to such H9C2 cells in a high-glucose environment.

Results: We found that STZ-treated mice had DCM, decreased cardiac function, necrotic cardiomyocytes, and limited proliferation. Western blot and immunofluorescence were used to detect the expression levels of various appropriate proteins in the myocardial tissue of mice and H9C2 cells. Compared to those in the control group, the expression levels of the apoptosis-related proteins cleaved caspase 3 and Bax were significantly higher in the experimental group, while the expression of the proliferation-related proteins proliferating cell nuclear antigen (PCNA) and CyclinD1 was significantly lower. In vivo and in vitro, the expression of TRPC6 and P-CaMKII increased in a high-glucose environment. However, addition of inhibitors to H9C2 cells in a high-glucose environment resulted in alleviation of both apoptosis and proliferation inhibition.

Conclusion: The inhibition of apoptosis and proliferation of cardiomyocytes in a high-glucose environment may be closely related to activation of the TRPC6/P-CaMKII pathway.

Keywords: Apoptosis; Diabetic cardiomyopathy; H9C2 cells; P-calmodulin dependent protein kinase II; Proliferation; Transient receptor potential channel 6.

Figure 1

Long axis ultrasound imaging and cardiac function measurement of the left ventricle. A: Left ventricular short axis view and cardiac function were measured in the control group and streptozotocin (STZ) injection group; B: Flow cytometry of reactive oxygen species in the control group and STZ injection group; C: HE, Masson, and periodic acid Schiff staining of diabetic cardiomyopathy and control mice. Scale bars = 50 μm. ^aP < 0.0001. STZ: Streptozotocin; ROS: Reactive oxygen species; PAS: Periodic acid Schiff.

Figure 2

Apoptosis of cardiomyocytes in diabetic cardiomyopathy mice. A: Apoptosis of diabetic cardiomyopathy (DCM) and control mice was detected using double staining of FITC-Annexin V and propidine iodide and the representative images of flow cytometry are presented. ^aP < 0.0001; B: Expression levels of apoptosis related proteins BAX, cleaved Caspase 3 (CC3), and Bcl2 in DCM mice and control mice. n = 5, unpaired t-test, ^bP < 0.01. n.s.: No statistical difference; C: Fluorescence intensity of CC3 in cardiac myocytes of DCM and control mice. Scale bars = 20 μm, ^bP < 0.01. DCM: Diabetic cardiomyopathy.

Figure 3

Proliferation of cardiomyocytes in diabetic cardiomyopathy mice. A: Cell cycle was detected by flow cytometry for the percentage of cells in each cell cycle of diabetic cardiomyopathy (DCM) and control mice. n = 5, unpaired t-test, ^aP < 0.001; B: The expression levels of cardiac cell cycle related proteins PCNA and CyclinD1 in DCM and control mice. n = 5, unpaired t-test, ^bP < 0.01; C: Fluorescence intensity of PCNA in cardiac myocytes of DCM and control mice. Scale bars = 20 μm, ^bP < 0.01. DCM: Diabetic cardiomyopathy.

Figure 4

Expression of transient receptor potential channel 6 and P-CAMKII proteins in the myocardium of diabetic cardiomyopathy mice. A: Expression levels of calcium channel protein transient receptor potential channel 6 (TRPC6) and P-CAMKII in cardiac myocytes of diabetic cardiomyopathy (DCM) and control mice. n = 5, unpaired t-test, ^aP < 0.01. n.s.: No statistical difference; B: Fluorescence intensity of TRPC6 in cardiac myocytes of DCM and control mice. Scale bars = 20 μm, ^bP < 0.001. DCM: Diabetic cardiomyopathy; TRPC6: Transient receptor potential channel 6.

Figure 5

Pathological and biochemical changes of H9C2 cardiomyocytes in each group. A: After high glucose induced injury of H9C2 cells, the effects of SAR7334 and KN-93 on the proliferation of H9C2 cells were observed by Cell Counting Kit-8 method. ^aP < 0.01, ^bP < 0.05; B: After high glucose induced injury of H9C2 cells, lactate dehydrogenase method was used to detect the effects of SAR and KN-93 on the mortality of H9C2 cells. n = 5, unpaired t-test, ^aP < 0.01, ^cP < 0.001; C: Reactive oxygen species fluorescence intensity of each group after treatment with fluorescent probe DCFH-DA. n = 5, unpaired t-test, scale bars = 20 μm, ^cP < 0.001, ^dP < 0.0001. HG: High-glucose; LG: Low-glucose.

Figure 6

Effects of SAR7334 and KN-93 on high-glucose-induced apoptosis of H9C2 cells. A: Apoptosis rate of H9C2 cells in each group was detected by flow cytometry. n = 5, unpaired t-test, ^aP < 0.0001; B: Effects of SAR7334 and KN-93 on the expression of apoptosis related proteins Bax, cleaved Caspase 3 (CC3), and BCl2 in H9C2 cells induced by high glucose. n = 5, unpaired t-test, ^bP < 0.001. n.s.: No statistical difference; C and D: Expression of apoptosis protein CC3 detected by immunofluorescence. n = 5, unpaired t-test, ^aP < 0.0001, ^bP < 0.001, ^cP < 0.01. Scale bars = 10 μm. HG: High-glucose; LG: Low-glucose.

Figure 7

Effects of SAR7334 and KN-93 on high-glucose-induced inhibition of H9C2 cell proliferation. A: Percentage of cells in each group in each period was detected by flow cytometry. n = 5, unpaired t-test, ^aP < 0.001, ^bP < 0.01; B: Effects of SAR7334 and KN-93 on the expression levels of cycle related proteins PCNA and CyclinD1 in H9C2 cells induced by high glucose. n = 5, unpaired t-test, ^aP < 0.001, ^bP < 0.01, ^cP < 0.0001. n.s.: No statistical difference; C and D: Expression of PCNA detected by immunofluorescence. n = 5, unpaired t-test, ^aP < 0.001, ^bP < 0.01, ^cP < 0.0001. Scale bars = 10 μm. HG: High-glucose; LG: Low-glucose.

Figure 8

Changes of expression of transient receptor potential channel 6 (TRPC6) and P-CaMKll under the intervention of SAR7334 and KN-93. A: Effects of SAR7334 and KN-93 on the expression of transient receptor potential channel 6 (TRPC6) and P-CaMKll in high glucose induced H9C2 cells. n = 5, unpaired t-test, ^aP < 0.001. n.s.: No statistical difference; B and C: Expression of TRPC6 protein detected by immunofluorescence. n = 5, unpaired t-test, ^bP < 0.0001. Scale bars = 10 μm. HG: High-glucose; LG: Low-glucose; TRPC6: Transient receptor potential channel 6.