LOXL2 silencing suppresses angiotensin II-induced cardiac hypertrophy through the EMT process and TGF-β1/Smad3/NF-κB pathway.

Figures

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  Figure 1. LOXL2 expression in Ang II-induced cardiomyocytes. Cultured H9c2 cells were treated by vehicle (Control) or Ang II (1 μM) for 12, 24, and 48 hr. A: mRNA expression level of LOXL2 in Ang II-induced H9c2 cells. B: Western blot was carried out to determine the protein expression level of LOXL2 in Ang II-induced H9c2 cells. C: Protein expression level of LOXL2 is increased by Ang II treatment in H9c2 cells. mRNA expressions of D: ANP, E: BNP, and F: β-MHC genes were evaluated by RT‐qPCR. All data are expressed as mean ± SEM, and the experiment was performed three times. *P<0.05, **P<0.01, and ***P<0.001 vs control group

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  Figure 2. SiLOXL2 inhibits Ang II‐induced cardiomyocyte hypertrophy. H9c2 cells were transfected with siLOXL2 or siNC and treated with Ang II (1 μM) for 48 hr. A: Representative images of cardiomyocyte morphology by immunofluorescent staining with α‐actinin (green) and DAPI (blue) (scale bar: 5 μm). B: Surface area of cardiomyocytes is quantified by measuring 100 cardiomyocytes from each group. C: RT‐qPCR is performed to evaluate the mRNA expressions of ANP, D: BNP, and E: β-MHC genes; GAPDH is used as the internal control. ***P<0.001 vs cardiomyocytes in the control; ###P<0.001 vs cardiomyocytes with Ang II

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  Figure 3. Effects of siLOXL2 on EMT markers in cardiomyocytes. Cultured H9c2 cells were transfected with siLOXL2 or siNC, and treated with Ang II (1 μM) for 48 hr. A: Western blot is performed to assess protein levels of EMT markers, and representative bands are shown. Bands are quantified by ImageJ software and show that siLOXL2 significantly attenuates the decrease in B: E-cadherin, and attenuates the increases in C: Vimentin, D: α-SMA, E: FSP, and F: Collagen 1A1 protein expressions. β-actin served as a loading control. ***P<0.001 vs cardiomyocytes in the control; ###P<0.001 vs cardiomyocytes with Ang II

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  Figure 4. Effect of siLOXL2 on the expression of TGF-β1/Smad3/NF-κB pathway and inflammatory response. H9c2 cells are transfected with siLOXL2 or siNC and incubated with Ang II for 48 hr. A: Western blot was performed and shows the representative bands. siTNC significantly attenuates the Ang II-induced increase in B: TGF-β1, C: p-Smad3, and D: p-NF-κB, but does not change the E: NF-κB protein level. β-actin served as a loading control. RT-qPCR is performed to detect the mRNA levels of F: IL-1β and G: TNF-α in H9c2 cells. ELISA was performed to detect the secreted H: IL-1β and I: TNF-α in the cell supernatant of H9c2 cells. ***P<0.001 vs cardiomyocytes in the control; ##P<0.01 and ###P<0.001 vs cardiomyocytes with Ang II