Thirty male Dahl rats and 40 SD rats (Charles River Laboratory Animal Technology Co., Ltd., China; license number: SCXK 2012-0001) at 6 weeks of age were randomly divided into the following groups including Dahl control group (Dahl + NS), Dahl high salt group (Dahl + HS), Dahl high salt + NaHS group (Dahl + HS + NaHS), SD control group (SD + NS), SD high salt group (SD + HS), SD high salt + sodium hydrosulfide (NaHS) group (SD + HS + NaHS) and SD high salt + hydroxylamine (HA) group (SD + HS + HA). Rats in Dahl + NS and SD + NS groups were given chow with 0.5% sodium chloride (NaCl) and 0.9% saline by intraperitoneal injection daily. Rats in Dahl + HS and SD + HS groups were given chow with 8% NaCl and 0.9% saline by intraperitoneal injection daily. Rats in the Dahl + HS + NaHS and SD + HS + NaHS groups were given chow with 8% NaCl and 90 μmol/kg NaHS by intraperitoneal injection daily (Shi et al., 2007). Rats in the SD + HS + HA group were given chow with 8% NaCl and 12.5 mg/kg HA (an inhibitor of CBS) per day (Han et al., 2005) and NaHS and HA solutions were freshly prepared with 0.9% saline daily. All rats were housed in Animal Center of Peking University First Hospital, freely drinking water and food throughout the whole experiment. The rats were kept at 25°C constant temperature and circadian rhythm of 12 h/12 h. The animal experiment was approved by the Experimental Animal Ethics Committee of Peking University First Hospital and strictly followed the regulations and guidelines of experimental animals of Peking University First Hospital.

At the end of the experiment, the body weight of conscious and quiet rats was measured. The rats were anesthetized by intraperitoneal injection of 25% urethane at 1.25 g/kg body weight. After the chest was opened, the hearts were removed rapidly and the heart weight was measured.

Appropriately 2 mm of cardiac cross-section tissue was taken, fixed in 4% paraformaldehyde and paraffin-embedded. Then, 5 μm of slice was taken for hematoxylin-eosin (HE) staining. Image acquisition and quantitative analysis of cardiomyocyte HE staining were performed by Leica Q550CW Image Processing and Analysis System. Each sample was randomly selected three fields from the left ventricular tissue, which were used to calculate the size of myocardial cells. The tissue samples of paraffin sections were put in xylene for 5 min, totally three times, then 100, 95, and 75% ethanol for 5 min separately, deionized water for 5 min, totally three times and then stained with Dio solution of 25 μmol/L overnight at 4°C. Fluorescence image acquisition and analysis were performed by using a laser scanning confocal microscope (Olympus, Japan). Three fields were randomly taken from each sample, and approximately 100 cells were quantitatively analyzed for cell size (Wu et al., 2012).

Total ribonucleic acid (RNA) was extracted from myocardial tissues by Trizol method. The purity and concentration of the total RNA were determined by the quantitative detection of nucleic acid protein. Two microgram of total RNA was used for complementary deoxyribonucleic acid (cDNA) synthesis by reverse transcription. cDNA synthesis conditions included 25 μL of reaction system for denaturation at 70°C for 5 min, and then at 42°C reaction for 60 min in PCR instrument. Twenty-five microliter of mixture was expanded in an ABI Prism 7300 instrument (Applied Biosystems, Foster, CA, USA). RT-PCR reaction conditions were set at 95°C pre-denaturation for 10 min, 95°C denaturation for 15 s, 60°C annealing for 1 min, and 72°C polymerization extension for 30 s, with a total of 40 cycles. β-actin was used as an internal reference for the calibration. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences were shown in Table 1. The ABI prism 7300 system software was for Ct and 2^-ΔΔt data analysis (Luo et al., 2013).

Myocardial tissues were ground into homogenate according to the mass volume ratio 1:10 (mg/μL) adding 1 × tissue lysate, and then the homogenate was centrifuged for 10 min at 4°C for supernatant. The supernatant was collected and added into the same volume of 2 × loading buffer at 100°C for 5 min. Each channel was added with equal amount of protein mixture for polyacrylamide gel electrophoresis, transferred into membrane. The primary antibodies including α-myosin heavy chain (α-MHC; 1:200, Santa Cruz Biotechnology, USA), β-MHC (1:200, Santa Cruz Biotechnology, USA), CBS (1:2000, Santa Cruz Biotechnology, USA), CSE (1:500, Sigma, USA), MPST (1:4000, Santa Cruz Biotechnology, USA), superoxide dismutase 1 (SOD1; 1:5000, Stressgen, USA), SOD2 (1:5000, Stressgen, USA), β-actin (1:2000, Santa Cruz Biotechnology, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000, Kangcheng, Shanghai, China) were added into membrane overnight at 4°C for incubation, and then washed by TTBS for 10 min/time of total of four times, with secondary antibodies for 1 h at room temperature for full incubation. AlphaImager gel imaging system was used to scan the protein band and measure the optical density of the protein band, and β-actin and GAPDH as internal reference protein were used to correct (Luo et al., 2013).

Appropriate amount of myocardial tissues was weighed and placed in 1.5 ml of EP tube, according to the mass volume ratio (1/10) with phosphate buffer saline (PBS) solution (pH7.2, 50 mmol/L), then myocardial tissues were ground into homogenate which was centrifuged at 12000 g for 10 min at 4°C for the supernatant, and still then the supernatant was measured to protein content. The H₂S content in the supernatant was determined using a Free Radical Detection Analyzer TBR4100 (World Precision Instruments, Shanghai, China) (Fox et al., 2012). At First, a 100 μm of ISO-H₂S-100 sensor (ISO-H₂S-100, WPI, Shanghai, China) was polarized in PBS buffer solution (pH 7.2, 50 mmol/L). The sensor could be calibrated as a stable reference current came out (usually 100–2000 pA). Approximately 10 mm of the sensor tip was immersed into 20 ml of PBS buffer solution (pH 7.2, 50 mmol/L) until a steady current appeared on the display. Six different concentrations of sodium sulfide solution (1, 2, 4, 8, 16, and 32 μmol/L) prepared in PBS buffer were successively detected by the detecting probe. After each sample added, the current output rapidly increased to the plateau. As long as the current reached the plateau, the next sample could be detected. The calibration curve was prepared from the signal output (pA) and the corresponding H₂S concentration (μM). The length of immersion was about 10 mm for each sample. The H₂S content in each sample could be calculated according to the calibration curve of pA-H₂S concentration.

Appropriate amount of myocardial tissues was put into PBS buffer (pH 7.2, 10 mmol/L) according to the mass ratio of 1:10 (mg/μl), and was fully ground into homogenate which was centrifuged at 12000 g for 10 min at 4°C for the supernatant. The activities of SOD, catalase (CAT), glutathione peroxidase (GSH-PX), and oxidized glutathione (GSSG) were determined by biochemical colorimetry. Glutathione (GSH), malondialdehyde (MDA), hydroxyl radical (⋅OH), carbon monoxide (CO), and total antioxidant capacity (T-AOC) were measured by biochemical colorimetry. The experimental procedure was strictly carried out according to the kit (Nanjing Jiancheng Biology Engineering Research Institute, China).

The data statistical analysis was performed using ABI SPSS 13.0 software. The results was expressed as mean ± standard error (mean ± SEM). The means among more than 2 groups were compared using one-way ANOVA. P < 0.05 was significant.

Compared with rats in Dahl + NS group, H₂S content of myocardial tissues was significantly decreased (P < 0.01; Figure 1A) with downregulated CBS mRNA and protein expression (P < 0.01; Figures 1B1,C1) and reduced CSE mRNA (P < 0.01; Figure 1B2), but CSE protein expression and MPST mRNA and protein expressions of myocardial tissues did not differ in Dahl + HS group rats (P > 0.05; Figures 1B3,C2,C3). There was no obvious difference in H₂S content and CBS, CSE and MPST mRNA and protein expressions of myocardial tissues among SD + NS group, SD + HS group and SD + HS + NaHS group rats (P > 0.05; Figures 2A–C). But, compared with SD rats, H₂S content and CBS protein expression of myocardial tissues were significantly reduced (P < 0.01; Figures 2A,C1), whereas CBS mRNA, CSE and MPST mRNA and protein expressions did not change in SD rats treated with HA (P > 0.05; Figures 2B,C2,C3).

Compared with rats in Dahl + NS group, those in Dahl + HS group showed marked myocardial hypertrophy including increased heart weight/body weight (P < 0.01; Figure 3A) and cardiomyocyte cross-sectional area (P < 0.01; Figures 3B1–B4,C1–C4), decreased α-MHC protein, but increased β-MHC protein (P < 0.01; Figures 3D1,D2). However, compared with rats in Dahl + HS group, those in Dahl + HS + NaHS group exhibited lessened myocardial hypertrophy characterized by decreased heart weight/body weight (P < 0.05; Figure 3A), reduced cardiomyocyte cross-sectional area (P < 0.01; Figures 3B1–B4,C1–C4), increased α-MHC protein, but decreased β-MHC protein (P < 0.05, P < 0.01; Figures 3D1,D2).

There was no statistical difference in myocardial structure, and α-MHC and β-MHC protein expressions of myocardial tissues of rats among SD + NS group, SD + HS group and SD + HS + NaHS group (P > 0.05; Figures 4A–D). However, compared with those in rats of SD + HS group, ratio of heart weight/body weight and cardiomyocyte cross-sectional area were increased, α-MHC protein was downregulated but β-MHC protein expression was upregulated in myocardial tissues of rats in SD + HS + HA group (P < 0.05, P < 0.01; Figures 4A–D).

Compared with rats of Dahl + NS group, the levels of T-AOC, CO and GSH, the activities of CAT, GSH-Px and SOD (P < 0.05, P < 0.01; Figures 5A1–A6) and protein expressions of SOD1 and SOD2 were decreased (P < 0.01; Figures 5B1,B2), but the contents of ⋅OH, MDA, and GSSG were obviously increased (P < 0.05, P < 0.01; Figures 5C1–C3) in the myocardial tissues of rats in Dahl + HS group. Compared with rats of Dahl + HS group, the levels of T-AOC, CO, and GSH, the activities of CAT, GSH-Px, and SOD (P < 0.05; Figures 5A1–A6), and protein expressions of SOD1 and SOD2 were significantly increased (P < 0.05, P < 0.01; Figures 5B1,B2), but the contents of ⋅OH, MDA, and GSSG were significantly decreased (P < 0.05; Figures 5C1–C3) in myocardial tissues of rats in Dahl + HS + NaHS group.

There was no significant difference in the levels of T-AOC, CO and GSH, the activities of CAT, GSH-Px and SOD, protein expressions of SOD1 and SOD2 (P > 0.05; Figures 6A1–A6,B1,B2) as well as the contents of ⋅OH, MDA, and GSSG of myocardial tissues (P > 0.05; Figures 6C1–C3) among SD + NS group, SD + HS group and SD + HS + NaHS group of rats. However, compared with those of SD + HS group, the levels of T-AOC, CO and GSH, the activities of CAT, GSH-Px and SOD as well as protein expressions of SOD1 and SOD2 were significantly decreased (P < 0.05, P < 0.01; Figures 6A1–A6,B1,B2), but the contents of ⋅OH, MDA, and GSSG were obviously increased (P < 0.05; Figures 6C1–C3) in the myocardial tissues of rats in SD + HS + HA group.