Molecular role of GATA binding protein 4 (GATA-4) in hyperglycemia-induced reduction of cardiac contractility

Abstract

Background: Diabetic cardiomyopathy, a diabetes-specific complication, refers to a disorder that eventually leads to left ventricular hypertrophy in addition to diastolic and systolic dysfunction. In recent studies, hyperglycemia-induced reactive oxygen species (ROS) in cardiomyocytes have been linked to diabetic cardiomyopathy. GATA binding protein 4 (GATA-4) regulates the expression of many cardio-structural genes including cardiac troponin-I (cTnI).

Methods: Streptozotocin-induced diabetic rats and H9c2 embryonic rat cardiomyocytes treated with a high concentration of glucose (a D-glucose concentration of 30 mM was used and cells were cultured for 24 hr) were used to examine the effect of hyperglycemia on GATA-4 accumulation in the nucleus. cTnI expression was found to be linked to cardiac tonic dysfunction, and we evaluated the expression levels of cTnI and GATA-4 by Western blot analysis.

Results: Cardiac output was lowered in STZ-induced diabetic rats. In addition, higher expressions of cardiac troponin I (cTnI) and phosphorylated GATA-4 were identified in these rats by Western blotting. The changes were reversed by treatment with insulin or phlorizin after correction of the blood sugar level. In H9c2 cells, ROS production owing to the high glucose concentration increased the expression of cTnI and GATA-4 phosphorylation. However, hyperglycemia failed to increase the expression of cTnI when GATA-4 was silenced by small interfering RNA (siRNA) in H9c2 cells. Otherwise, activation of ERK is known to be a signal for phosphorylation of serine105 in GATA-4 to increase the DNA binding ability of this transcription factor. Moreover, GSK3β could directly interact with GATA-4 to cause GATA-4 to be exported from the nucleus. GATA-4 nuclear translocation and GSK3β ser9 phosphorylation were both elevated by a high glucose concentration in H9c2 cells. These changes were reversed by tiron (ROS scavenger), PD98059 (MEK/ERK inhibitor), or siRNA of GATA-4. Cell contractility measurement also indicated that the high glucose concentration decreased the contractility of H9c2 cells, and this was reduced by siRNA of GATA-4.

Conclusions: Hyperglycemia can cause systolic dysfunction and a higher expression of cTnI in cardiomyocytes through ROS, enhancing MEK/ERK-induced GATA-4 phosphorylation and accumulation in the cell nucleus.

Figure 1

Cardiac output and cardiac GATA-4 phosphorylation and troponin I expression in the heart. Comparison of streptozotocin-induced diabetic rats (STZ) and normal (Wistar) rats. Each column shows the mean ± SEM (n = 6). ***P < 0.001 (A). STZ diabetic rats were treated with insulin or phlorizin at a dose sufficient to correct the blood sugar level for 4 days. Then, rats were sacrificed and nuclear GATA-4 phosphorylation (B) and troponin I expression (C) were detected by Western blot analysis. The results are presented as the mean ± SEM (n = 6 per group). *P < 0.05 and **P < 0.01 as compared with the STZ rats.

Figure 2

Effects of tiron on ROS generation in neonatal rat cardiomyocytes and H9c2 cells. DHE staining was used to visualize the intracellular ROS and lucigenin assays were used to quantify the generation of superoxide in neonatal rat cardiomyocytes (A) and H9c2 cells (B). *P < 0.05 and **P < 0.01 compared with the control; ^#P < 0.05 and ^##P < 0.01 compared with HG.

Figure 3

GATA-4 and cardiac troponin I expression in neonatal rat cardiomyocytes and H9c2 cells. Cell nuclear fractions were isolated for Western blot analysis to detect the phosphorylation level of GATA-4 and troponin I in neonatal rat cardiomyocytes (A) and H9c2 cells (B). All values are expressed as the mean ± SEM (n = 4). **P < 0.01 and *** P < 0.001 as compared with high glucose-treated H9c2 cells.

Figure 4

Effects of MAPK inhibitors on the level of p-GATA-4 and cardiac cTnI expression. H9c2 cells were incubated with SB203580 (25 mmol/L) or PD98059 (20 mmol/L) for 30 min before exposure to 30 mmol/L glucose. Then, nuclear fractions were isolated for Western blot analysis of GATA-4 phosphorylation (A) and cardiac troponin I expression (B). In another group, H9c2 cells were treated with 1 mmol/L phenylephrine (PE) in normal medium for 24 hr. Western blotting analysis was then used to estimate the effect of PD98059 on the expression of troponin I induced by PE (C). The results are presented as the mean ± SEM (n = 3-5 per group). *P < 0.05, **P < 0.01 as compared with the high glucose-treated group.

Figure 5

Effects of GATA-4-specific RNAi on GATA-4 and cTnI expression in H9c2 cells. H9c2 cells were transfected with siRNA specific to GATA-4 (RNAi) or scrambled RNA (Scr) for 24 hr and then exposed to 30 mmol/L glucose for another 24 hr. Whole cell lysates were isolated for Western blot analysis of GATA-4 (A) and troponin I (B) protein expression. H9c2 cells cultured in regular medium were used as the control (Con). The results are presented as the mean ± SEM (n = 3-5 per group). *P < 0.05, **P < 0.01 as compared with the high glucose-treated group.

Figure 6

Effect of MEK/ERK inhibitor on GSK3β phosphorylation and GATA-4 translocation in neonatal rat cardiacmyocytes and H9c2 cells. H9c2 cells were exposed to 30 mmol/L glucose for 24 hrs, and subsequently the nuclear fraction and cytosolic fraction were isolated for Western blot analysis of GATA-4 expression and GSK3β phosphorylation (A and B). Changes in GATA-4 localization were also measured using a fluorescent microscope. Counter staining of the cell nucleus was achieved using DAPI (4', 6-diamidino-2-phenylindole) (A and D). H9c2 cells were incubated with PD98059 (20 mmol/L) for 30 min and subsequently exposed to the same glucose concentration (30 mmol/L) for 24 hr. The nuclear fractions were then isolated for Western blot analysis to measure phosphorylation of GSK3β (B). Result of western blot analysis in neonatal rat cardiomyocytes treated with HG at various doses(C). The results are presented as the mean ± SEM (n = 3 per group). *P < 0.05 as compared with the high glucose-treated group.

Figure 7

Effects of GATA-4 specific siRNA on phenylephrine (PE)-induced cell contraction in H9c2 cells. The high glucose-treated H9c2 cells were incubated with siRNA-GATA-4 (Si) or scrambled RNA as a control (Scramble) for 24 hr before PE challenge and then their planar surface area was measured. The upper panel (A) shows a representative change of cells in each group. The lower panel (B) expresses the differences between the four groups as the mean ± SEM (n = 6 per group). The high glucose-treated group differed markedly from the control incubated in normal medium (***P < 0.001). The high glucose-treated GATA-4 RNAi group differed markedly from the scrambled RNA-treated group treated with a high glucose concentration (^##P < 0.01, ^###P < 0.001).

Figure 8

Possible mechanism of hyperglycemia in change of cardiac troponin I expression in type-1 diabetic rats. Hyperglycemia-induced ROS activates the MEK/ERK pathway to increase GATA-4 phosphorylation and nuclear translocation. Also, MEK/ERK activation causes GSK3β phosphorylation, lowering the export of GATA-4 and leading to GATA-4 preservation in the nucleus, which finally leads to an increase in the expression level of cTnI.