Angiotensin II prompts heart cell apoptosis via AT1 receptor-augmented phosphatase and tensin homolog and miR-320-3p functions to enhance suppression of the IGF1R-PI3K-AKT survival pathway

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  FIGURE 1. Angiotensin II-stimulated AT1 receptor expression to suppress the IGF1R survival pathway in H9c2 cardiomyoblasts. (a) Angiotensin II significantly enhanced AT1 receptor expression in H9c2 cardiomyoblasts to suppress the IGF1R-PI3K/AKT pathways and inducing cleavage of caspase 8 and caspase 3. Angiotensin II: 100 nmol/l, isoproterenol: 50 nmol/l, and lipopolysaccharides: 2 μg/ml (b) angiotensin II dose-dependent significantly increased expression levels of AT1 receptor and cleavage of caspase 3, with concomitant downregulation of GF1R-PI3K/AKT signaling pathways. (c) The treatment of angiotensin II affected AT1 receptor to downregulate the IGF1R pathway in a time-dependent manner. The prolonged treatment time attenuated AKT expression levels and upregulated cleaved caspase 8 and caspase 3 to activate cardiac cell apoptosis. Angiotensin II: 100 nmol/l. The protein expression levels were normalized to internal control β-actin. The results are expressed as protein fold change in treated groups compared with the control. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent…

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  FIGURE 2. Phosphatase and tensin homolog was upregulated by angiotensin II in H9c2 cardiomyoblasts. (a) Angiotensin II, isoproterenol, and lipopolysaccharides induced an increase in phosphatase and tensin homolog expression levels compared with control untreated H9c2 cardiomyoblasts. Angiotensin II: 100 nmol/l, isoproterenol: 50 nmol/l, and lipopolysaccharides: 2 μg/ml (b), angiotensin II treatment dose-dependently and (c), time-dependently upregulated phosphatase and tensin homolog expression levels. (d) SF1670 (phosphatase and tensin homolog Inhibitor) and losartan (AT1 receptor inhibitor) combination treatment with angiotensin II reversed angiotensin II-induced inhibition of Akt phosphorylation in H9c2 cardiomyoblasts. Angiotensin II: 200 nmol/l, SF1670: 2.5 μol/l, and losartan: 1.5 μmol/l. The protein expression levels were normalized to internal control β-actin (a, b and c) or GAPDH (d). Results are expressed as protein fold change in treated groups compared with the control. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent experiments (ns: not significant, ∗P < 0.05, ∗∗P <…

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  FIGURE 3. Inhibition of the angiotensin II-AT1 receptor axis activated the IGF1R survival pathway to reduce apoptosis in angiotensin II-exposed H9c2 cardiomyoblasts. Losartan (an AT1 receptor inhibitor) reduced the expression levels of AT1 receptor and inhibited angiotensin II-AT1 receptor signaling. Downstream IGF1R was upregulated, which further enhanced the activity of Akt and inhibited apoptotic markers, including caspase 8 and caspase 3. The inhibition of the angiotensin II-AT1 receptor axis with losartan could be dose-dependent. The protein expression levels were normalized to internal control β-actin. The results are expressed as protein fold change in treated groups compared with the control. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent experiments (ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

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  FIGURE 4. Angiotensin II upregulated miR-320-3p expression in H9c2 cardiomyoblasts. The miR-320-3p fold-change qPCR quantification in angiotensin II and losarten-treated H9c2 cardiomyoblasts. (a) The expression of miR-320-3p was increased in angiotensin II-exposed H9c2 cardiomyoblasts. (b) The increase in mir-320-3p was even more significant over time in angiotensin II-exposed H9c2 cardiomyoblasts. (c) The increased level of mir-320-3p was reduced by losartan (AT1 receptor inhibitor). Con: control group. The expression levels of mir-320-3p were normalized to U6 internal control expression level. The results are expressed mir-320-3p fold change in treated groups compared with the control. Data are represented as mean ± SD from three independent experiments (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

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  FIGURE 5. The PI3K-AKT pathway in angiotensin II-exposed H9c2 cardiomyoblasts was regulated by miR-320-3p. (a) mir-320-3p overexpression significantly reduced the phosphorylation of Akt, via a mir-320 mimic, in H9c2 cardiomyoblasts. (b) The expression of PI3K and Akt was downregulated when H9c2 cardiomyoblasts were co-treated with angiotensin II and a mir-320 mimic. (c) mir-320-3p inhibition, via a mir-320 inhibitor, increased the phosphorylation of Akt in H9c2 cardiomyoblasts. (d) Otherwise, the mir-320 inhibitor enhanced the phosphorylation of PI3K and AKT to reduce apoptosis in angiotensin II-exposed H9c2 cardiomyoblasts. The protein expression levels were normalized to internal control GAPDH (a, c) or β-actin (b, d). The results are expressed as protein fold change in treated groups compared with the control. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent experiments (ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

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  FIGURE 6. The apoptosis induced by angiotensin II in H9C2 cells was enhanced by miR-320-3p. angiotensin II-induced apoptosis was confirmed by TUNEL staining assay. The miR-320-3p overexpression significantly enhanced the number of TUNEL-positive cells. Consequently, reduced expression of mir-320-3p by mir-320-3p inhibitor reversed the apoptosis in angiotensin II-exposed H9C2 cells. Representative images are shown. Scale bars: 50 μm.

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  FIGURE 7. The activation of AKT in angiotensin II-exposed neonatal rat cardiomyocytes was affected by miR-320-3p. (a) mir-320-3p overexpression, via a mir-320 mimic, significantly reduced the phosphorylation of Akt in angiotensin II-exposed neonatal rat cardiomyocytes. (b) mir-320-3p inhibition, via a mir-320 inhibitor, increased the phosphorylation of Akt in angiotensin II-exposed neonatal rat cardiomyocytes. The protein expression levels were normalized to internal control GAPDH. The results are expressed as protein fold change in treated groups compared with the control. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent experiments (ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

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  FIGURE 8. The IGF1R-PI3K-AKT survival pathway was reduced in the hearts of hypertensive rats. (a) All groups showed decreased expression of activated IGF1R and AKT with age, but the activation of IGF1R and AKT in the hearts of spontaneously hypertensive rats was significantly lower than that in Wistar-Kyoto rats. 5W: Wistar-Kyoto rats (5 months old); 5S: spontaneously hypertensive rats (5 months old); 8W: Wistar-Kyoto rats (8 months old); 8S: spontaneously hypertensive rats (8 months old); 24W: Wistar-Kyoto rats (24 months old); 24S: (24 months old. The protein expression levels were normalized to internal control β-actin. The results are expressed as protein fold change in treated groups compared with the 5-months-old Wistar-Kyoto rats. Analysis of variance followed by the Tukey test was used to analyze the differences between the groups. Data are represented as mean ± SD from three independent experiments (ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (b) The phosphatase and tensin homolog was detected by immunohistochemistry, the phosphatase and tensin homolog expression level was increased in spontaneously hypertensive rats (24 months old) compared with Wistar-Kyoto rats…

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  FIGURE 9. Schematic diagram of IGF1R-PI3K-AKT downregulation mediated by angiotensin II-induced AT1 receptor, phosphatase and tensin homolog, and miR-320-3p in hypertension-associated heart damage.