Nitric Oxide Alleviated High Salt-Induced Cardiomyocyte Apoptosis and Autophagy Independent of Blood Pressure in Rats

Abstract

The present study aimed to explore whether high-salt diet (HSD) could cause cardiac damage independent of blood pressure, and whether nitric oxide (NO) could alleviate high-salt-induced cardiomyocyte apoptosis and autophagy in rats. The rats received 8% HSD in vivo. H9C2 cells or primary neonatal rat cardiomyocytes (NRCM) were treated with sodium chloride (NaCl) in vitro. The levels of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2, LC3 II/LC3 I, Beclin-1 and autophagy related 7 (ATG7) were increased in the heart of HSD rats with hypertension (HTN), and in hypertension-prone (HP) and hypertension-resistant (HR) rats. Middle and high doses (50 and 100 mM) of NaCl increased the level of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2, LC3 II/LC3 I, Beclin-1, and ATG7 in H9C2 cells and NRCM. The endothelial NO synthase (eNOS) level was increased, but p-eNOS level was reduced in the heart of HSD rats and H9C2 cells treated with 100 mM NaCl. The level of NO was reduced in the serum and heart of HSD rats. NO donor sodium nitroprusside (SNP) reversed the increases of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2 induced by NaCl (100 mM) in H9C2 cells and NRCM. SNP treatment attenuated the increases of cleaved-caspase 3/caspase 3, Bax/Bcl2, LC3 II/LC3 I, Beclin-1, and ATG7 in the heart, but had no effect on the blood pressure of HSD rats with HR. These results demonstrated that HSD enhanced cardiac damage independently of blood pressure. Exogenous NO supplementarity could alleviate the high salt-induced apoptosis and autophagy in cardiomyocytes.

Keywords: apoptosis; autophagy; cardiomyocytes; high-salt diet; nitric oxide; sodium nitroprusside.

FIGURE 1

Effects of high-salt diet (HSD) on body weight and blood pressure in rats. (A) The body weight in the HSD groups with hypertension-resistant (HR), hypertension-prone (HP), and hypertension (HTN) was less than that in the control diet (CD) group. (B–D) The SBP, DBP, and MAP in HTN were higher than that in CD, HR, and HP groups. The results are expressed as mean ± SEM; n = 10 in CD group, n = 14 in HR and HP group, and n = 17 in HTN group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^****p < 0.0001.

FIGURE 2

Effects of high-salt diet (HSD) on apoptosis and autophagy in the heart of hypertension-resistant (HR), hypertension-prone (HP), and hypertension (HTN) rats. The expression levels of cleaved-caspase 3/caspase 3 (A,B), cleaved-caspase 8/caspase 8 (A,C), Bax/Bcl2 (A,D), LC3 II/LC3 I (A,E), Beclin-1 (F) and autophagy related 7 (ATG7) (G) were increased in the heart of HSD rats with HR, HP and HTN. The results are expressed as mean ± SEM; n = 6 in each group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Effects of different doses of sodium chloride (NaCl) on apoptosis and autophagy in H9C2 cells. Middle (50 mM) and high (100 mM) doses of NaCl increased the levels of cleaved-caspase 3/caspase 3 (A,B), cleaved-caspase 8/caspase 8 (A,C), Bax/Bcl2 (A,D), LC3 II/LC3 I (A,E), Beclin-1 (F), and autophagy related 7 (ATG7) (G) in H9C2 cells. The results are expressed as mean ± SEM; n = 6 in each group. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Levels of endothelial nitric oxide synthase (eNOS), p-eNOS, inducible NOS (iNOS), and nitric oxide (NO). (A) The level of eNOS was higher, but p-eNOS was lower in H9C2 cells treated with middle (50 mM) and high (100 mM) doses of sodium chloride (NaCl). (B) The expression of eNOS was increased, but p-eNOS was reduced in the heart of high-salt diet (HSD) rats with hypertension-resistant (HR). (C) The level of iNOS was higher in H9C2 cells treated with middle (50 mM) and high (100 mM) doses of NaCl. (D) The level of iNOS was increased in the heart of HSD rats with HR. (E) NO level was reduced in the heart of HSD rats with HR. (F) NO level was reduced in the serum, but was increased in the 24-h urine of HSD rats with HR. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

Effects of different doses of nitric oxide donor sodium nitroprusside (SNP) on NaCl-induced apoptosis and autophagy in H9C2 cells. (A–I), SNP attenuated the increases in the levels of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2, LC3 II/LC3 I, Beclin-1, and autophagy related 7 (ATG7), and enhanced the decrease of p-endothelial nitric oxide synthase (eNOS) induced by NaCl (100 mM) in H9C2 cells. (J) The increase of TUNEL-positive cell number was inhibited by high dose of SNP (100 μM), but not middle (10 μM) or low dose (1 μM) of SNP. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 6

Effects of nitric oxide donor sodium nitroprusside (SNP) on apoptosis and autophagy induced by three doses of sodium chloride (NaCl) in H9C2 cells. (A–I) SNP (100 μM) attenuated the increases in the levels of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, and Bax/Bcl2 induced by NaCl (50 or 100 mM) in H9C2 cells. (F) The increases of TUNEL-positive cell numbers induced by three doses of NaCl in the H9C2 were attenuated by SNP (100 μM) treatment. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 7

Effects of nitric oxide donor sodium nitroprusside (SNP) on apoptosis and autophagy induced by three doses of sodium chloride (NaCl) in primary neonatal rat cardiomyocytes (NRCM). SNP (100 μM) attenuated the increases in the levels of cleaved-caspase 3/caspase 3 (A,B), cleaved-caspase 8/caspase 8 (A,C), Bax/Bcl2 (A,D), LC3 II/LC3 I (A,E), Beclin-1 (H), and autophagy related 7 (ATG7) (I), and enhanced the decrease of p-endothelial nitric oxide synthase (eNOS) induced by NaCl (50 or 100 mM) in NRCM (F,G). The increases of TUNEL-positive cell numbers induced by three doses of NaCl in the NRCM were attenuated by SNP (100 μM) treatment (J). The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 8

Effects of nitric oxide donor sodium nitroprusside (SNP) on HSD-induced apoptosis and autophagy in the heart of hypertension-resistant (HR) rats. (A–H) SNP attenuated the increases in cleaved-caspase 3/caspase 3, Bax/Bcl2, Beclin-1, and autophagy related 7 (ATG7), and enhanced the decrease of p-endothelial nitric oxide synthase (eNOS) induced by HSD in the heart of rats. (I) The increase of TUNEL-positive cell number was attenuated by SNP (25 μg/kg) treatment. (J) The decrease of nitric oxide (NO) in the heart of HSD rats with HR was increased by SNP administration. (K) SNP had no effects on the blood pressure of HR rats. The results are expressed as mean ± SEM. n = 6 for each group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.