The role of angiopoietin-like protein 4 in phenylephrine-induced cardiomyocyte hypertrophy

Figure 1. Effect of AB or PE on ANGPTL4 expression in cardiomyocytes (A–C) Representative images of the mouse heart at 3 days, 1 week and 4 weeks after AB or sham surgery. (D–F) The effect of AB on ANP mRNA expression was determined by q-PCR, and GAPDH was used as an internal control. (G–I) Quantitative analysis of the heart/body weight of mice at 3 days, 1 week and 4 weeks after aortic banding or sham surgery. (J) Western blots showing ANGPTL4 and GAPDH expression in mouse hearts at the indicated time after sham or AB surgery, and quantitative analysis of the above blots. (K) Western blots showing ANGPTL4 and GAPDH expression in cultured cardiomyocytes after vehicle or PE incubation for the indicated time, and quantitative analysis of the above blots. GAPDH was used as an internal control. *P<0.05 versus the corresponding control group. **P<0.01 versus the corresponding control group. The results represent three to five independent experiments; n = 3–5.

Figure 2. Effects of ANGPTL4 knockdown and recombinant ANGPTL4 treatment on PE-induced cardiomyocyte hypertrophy and the genes (PPARα, CPT-1 and CD36) involved in the regulation of FAO Cardiomyocytes were transfected with siRNA and incubated with or without PE for 24 h in serum-free medium. (A) The siRNA-mediated knockdown of ANGPTL4 was confirmed by q-PCR. (B) The effect of siRNA-mediated knockdown of ANGPTL4 on ANP mRNA expression was determined by q-PCR, and GAPDH was used as an internal control. (C) The effect of silencing ANGPTL4 on the cell surface area. After siRNA transfection, cardiomyocytes were treated with or without PE for 24 h. (D and G) Cardiomyocytes were stained with troponin I, and the nuclei were stained with DAPI. (E) The effect of recombinant ANGPTL4 on ANP mRNA expression was detected by q-PCR. (F) The effect of recombinant ANGPTL4 on the cell surface area. After siRNA transfection, the expression levels of PPARα (H), CPT-1 (I) and CD36 (J) were detected by q-PCR, and GAPDH was used as an internal control. *P<0.05 versus the corresponding control group; NS indicates no significance versus the corresponding control group. Each of the experiments was repeated four to…

Figure 3. Effect of ANGPTL4 knockdown on MAPK signaling (A) The MAPK bands were detected using Western blot analysis after PE incubation for 30 min. Quantitative analysis of phosphorylated and total ERK1/2 (B), JNK1/2 (C) and p38 (D). *P<0.05 versus the corresponding control group. NS indicates no significance versus the corresponding control group. Each of the experiments was repeated six times; n=6.

Figure 4. Effect of JNK inhibitor on ANGPTL4-mediated cardiomyocyte hypertrophy JNK inhibitor (SP600125) was added to cardiomyocytes 1 h prior to treatment with siRNA followed by treatment with or without PE for 24 h. (A) The effect of JNK inhibitor and siRNA on ANP mRNA expression was determined by q-PCR, and GAPDH was used as an internal control. (B) The effect of JNK inhibitor and ANGPTL4 knockdown on the cell surface area. (C) Cardiomyocytes were stained with troponin I, and the nuclei were stained with DAPI. (D) The expression levels of PPARα were detected by q-PCR; GAPDH was used as an internal control. (E) The effect of JNK inhibitor and ANGPTL4 knockdown on ANGPTL4 mRNA expression was detected by q-PCR. *P<0.05 versus the corresponding control group. **P<0.01 versus the corresponding control group. NS indicates no significance versus the corresponding control group. Each of the experiments was repeated three to six times; n=3–6.

Figure 5. Effect of JNK inhibitor on MAPK signaling following ANGPTL4 knockdown JNK inhibitor (SP600125) was added to cardiomyocytes 1 h prior to treatment with siRNA followed by treatment with or without PE for 24 h. (A) The MAPK bands were detected using Western blot analysis after PE incubation for 30 min. Quantitative analysis of phosphorylated and total JNK1/2 (B), ERK1/2 (C) and p38 (D). (E–G) The effect of AB on PPARα mRNA expression was determined by q-PCR, and GAPDH was used as an internal control. (H–J) The JNK1/2 bands were detected using Western blot analysis after AB surgery. (K–M) Quantitative analysis of phosphorylated and total JNK1/2. *P<0.05 versus the corresponding control group. NS indicates no significance versus the corresponding control group. Each of the experiments was repeated three to seven times; n=3–7.