Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid

Figure 2

RIPK1/RIPK3 expressions are significantly increased in cardiomyocytes with PA stimulation. (a) The increased gene expression of RIPK1/RIPK3 was inhibited by Nec-1 (10 nM) in NCMs (). (b) The increased protein level of RIPK1/RIPK3 in NCMs was downregulated by Nec-1 (10 nM) (). (c) Immunofluorescence for RIPK1 and RIPK3 in H9c2 cells. Note the higher immunoreactivity for RIPK1/RIPK3 in H9c2 cells (200x) with PA stimulation. The green fluorescence indicated RIPK1 staining, the red fluorescence indicated RIPK3 staining, and the blue fluorescence indicated the cell nucleus stained by DAPI. (d) Quantitative analysis of fluorescent microscopy images (c) (). (e) Transmission electron microscopy images of H9c2 cells treated with PA for 24 h showed lipid deposition within the cells. Necrotic morphology was observed including swollen mitochondria, cytoplasmic clearing, and membrane damage (M: mitochondrion; N: nucleus; L: lipid droplet; C: cytoplasmic clearing. Scale bars: 2 μm). (f) Treating the H9c2 cells with Nec-1 (10 nM), a specific necroptosis inhibitor, the increased gene levels of ANP, BNP, α-MHC, and β-MHC were downregulated via real-time PCR (). (g) After transfection with siRIPK1 or siRIPK3 (50 nM) in H9c2 cells, the PA-induced mRNA expression of RIPK1 or RIPK3 was significantly suppressed ( in each group). (h) After transfection with siRIPK1 or siRIPK3 (50 nM) in H9c2 cells, the PA-induced protein expression of RIPK1 or RIPK3 was significantly suppressed (). (i) Accordingly, silenced RIPK1 with siRIPK1 (siR1) or silenced RIPK3 (siR3) with siRIPK3 significantly inhibited both basal and PA-induced ANP and BNP gene expression in H9c2 cells (), as evaluated by quantitative RT-PCR. Data in (a), (b), (d), (f), (g), (h), and (i) are expressed as mean ± SD; indicates .

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