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

Abstract

Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

Figure 1

PA-induced hypertrophy in cardiomyocytes. (a) H9c2 cells were stained by Oil Red O dye. (1) Control (without any treatment). (2) Stimulation with PA (200 μM). (3) Pretreatment with Nec-1 (10 nM), n = 3. The result of Oil Red O⁺ (%population) indicated that lipid accumulation was induced in PA stimulation group in H9c2 cells, and the PA-induced intracellular accumulation of neutral lipids in H9c2 cells decreased in PA + Nec-1 group. (b) Gene expression of ANP, BNP, α-MHC, and β-MHC in H9c2 cells was induced by PA, but not OA, n = 3. (c) Gene expression of ANP and BNP was upregulated in NCMs, n = 4. (d) Fluorescence microscopy observed the increased H9c2 cells surface area of F-actin staining in PA group, which was suppressed by Nec-1 (10 nM), according to the semiquantitative results by ImageJ software, n = 3. The red fluorescence indicated cytoskeleton stained by rhodamine phalloidin and the blue fluorescence indicated the cell nucleus stained by DAPI. Data in (a), (b), (c), and (d) are expressed as mean ± SD, ∗ indicates p < 0.05 compared to control treatment, and # indicates p < 0.05 compared to PA treatment.

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 (n = 3). (b) The increased protein level of RIPK1/RIPK3 in NCMs was downregulated by Nec-1 (10 nM) (n = 4). (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) (n = 3). (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 (n = 3). (g) After transfection with siRIPK1 or siRIPK3 (50 nM) in H9c2 cells, the PA-induced mRNA expression of RIPK1 or RIPK3 was significantly suppressed (n = 4 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 (n = 3). (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 (n = 3), as evaluated by quantitative RT-PCR. Data in (a), (b), (d), (f), (g), (h), and (i) are expressed as mean ± SD; ∗ indicates p < 0.05.

Figure 3

There is a crosstalk between ER stress and necroptosis in PA-induced cardiomyocyte hypertrophy. (a) Treating the NCMs with PA (200 μM) and OA (250 μM), the mRNA expressions of GRP78, ATF6, and CHOP were increased in PA group, not OA group, n = 4. (b) Pretreating the H9c2 cells with pravastatin (10 μM) and effectively blocking the upregulated mRNA expression of GRP78, ATF6, and CHOP (ER stress markers) induced by PA and thapsigargin (100 nM) (an ER stress agonists) via real-time PCR, n = 3. (c) Pretreating the H9c2 cells with pravastatin (10 μM) and also effectively blocking the upregulated mRNA expression of RIPK1/RIPK3 induced by PA and thapsigargin via real-time PCR, n = 3. (d) The increased protein level of GRP78 and CRT in NCMs was downregulated by pravastatin (10 μM) (western blot), n = 3. Data in (a), (b), (c), and (d) are expressed as mean ± SD, ∗ indicates p < 0.05 compared to control treatment, and # indicates p < 0.05 compared to PA treatment.

Figure 4

AKT/mTOR mediates necroptosis in the PA-induced cardiomyocyte hypertrophy. (a) Pretreating the NCMs with rapamycin (1 μM), the mRNA level of ANP and BNP was decreased, n = 4. (b) Pretreating the NCMs with Nec-1 (10 nM), the upregulated Ser473 of AKT and Ser2481 of mTOR phosphorylation by PA stimulation (western blot) was inhibited. (c) Quantitative analysis of western blots (b), n = 3. (d) Pretreating the NCMs with rapamycin (1 μM) had no effect on the upregulated RIPK1 by PA stimulation via western blot, n = 6. Data in (a), (c), and (d) are expressed as mean ± SD, ∗ indicates p < 0.05 compared to control treatment, and # indicates p < 0.05 compared to PA treatment.

Figure 5

Schematic illustration of the signaling pathways of palmitic acid-induced cardiomyocyte hypertrophy.