A total of seven-week-old male SHR and Wistar Kyoto rats (WKY) weighing 200-250 g were obtained from Shanghai Laboratory Animal Center of the Chinese Academy of Science (Shanghai, China), and received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. no. 85-23, revised 1996), which was approved by the Institutional Ethics Review Board of the Characteristic Medical Center of the Chinese People's Armed Police Forces (Tianjin, China; approval number: 2012-0005). Rats were maintained in a 12/12-h dark/light cycle in air-conditioned rooms (23±1˚C, 55±5% humidity) and were acclimated to the local conditions for 1 week before the study. At 8 weeks of age, rats were divided randomly (n=12 for each group), and then given either a low-salt (LS; 0.5% NaCl) or HS diet (8% NaCl) for 8, 12 or 16 weeks. All rats were permitted free access to chow and tap water.

A total of 8, 12 and 16 weeks after dietary intervention, the rats were anesthetized (sodium pentobarbital, 60 mg/kg, intraperitoneally) for invasive hemodynamics using a 2F high fidelity micro-tip pressure catheter (SPR-320; Millar Instruments Ltd.) to measure systolic and diastolic BP (SBP and DBP), LV end-diastolic pressure (LVEDP), maximal slope of systolic pressure increment (+dP/dt_max), and diastolic pressure decrement (-dP/dt_min) as previously described by the authors (12).

Animals were euthanized with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneally) after in vivo hemodynamic measurement, and then were rapidly perfused via inferior vena cava with precooled physiological saline for 5 min. The hearts were quickly removed, sectioned, and prepared for paraffin embedding and cryo-section. Routine staining techniques in paraffin embedded tissue (7-µm) included hematoxylin and eosin (H&E) staining (25˚C, 15 min) and Masson's trichrome staining for evaluating interstitial collagen deposition. Tetraethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin (Invitrogen; Thermo Fisher Scientific, Inc.) plus 4,6-diamidino-2-phenylindole (DAPI; 5 mg/ml; Vector Laboratories, Inc.) staining was used to measure cardiomyocyte cross-sectional area, which was examined with a fluorescent microscope (80i; Nikon Corporation) as previously described by the authors (13). All image analysis was performed in a blinded manner by Image Pro Plus (version 4.5; Media Cybernetics, Inc.).

For detecting lipid droplet, Oil Red O staining was used in cryo-sectioned heart tissue (10 µm), and the sections were fixed with 10% buffered formalin (25˚C, 24 h) and stained with Oil Red O working solution (25˚C, 30 min). Hematoxylin was used for counterstaining. The perirenal adipose tissue was removed and smeared over a slide serving as the positive controls. For detecting sulfated proteoglycans, paraffin-embedded sections were stained with toluidine blue. Alcian blue/periodic acid-Schiff (AB-PAS) was used in paraffin-embedded sections to detect acidic sulfated mucins (AB positive), O-glycosides (PAS positive) and sialic acid (PAS positive) as previously described (14).

Transmission electron microscopy was performed for evaluating myocardial ultrastructure. Briefly, the samples from LV were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2; 4˚C, 24 h), post-fixed in 1.0% OsO4, dehydrated in alcohol and acetone solution, embedded in Epon812, sectioned with LKB ultramicrotome, and stained with uranyl acetate followed by lead citrate, then observed with a transmission electron microscope.

A total of 8 weeks after dietary intervention, when LV hemodynamic measurement was finished, rat hearts were harvested. The blood was collected from superior vena cava for measurement of plasma [Na⁺], [K⁺] and [Cl^-]. The right ventricle was dissected away, and the left ventricle was weighted to determine the wet weight (WW). Then the hearts were desiccated at 90˚C for 72 h and their dry weights (DW) were determined. Because the weight was unchanged with further drying, the difference between WW and DW was considered as tissue water content. The tissues were then ashed at 200˚C, 400˚C for 24 h at each temperature level and 600˚C for a further 48 h and then were dissolved in 20 ml 10% HNO₃. [Na⁺] and [K⁺] were measured by inductively-coupled plasma emission spectrometer (ICP, IRIS Intrepid II XSP, Thermo Electron Corporation). [Cl^-] was measured by titration with 0.1 N silver nitrate (15).

H9c2, a rat ventricular myoblast cell line (Shanghai Cell Bank, Chinese Academy of Science) was maintained at 37˚C and 5% CO₂ in Dulbecco's modified Eagle's medium (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% glutamine. H9c2 cells were seeded onto six-well plates and incubated with culture media containing NaCl (50 mM) or mannitol (100 mM), which yielded hypertonic state, equaling 400 mOsm/l, and treated with or without ROS scavenger N-acetylcysteine (NAC; 0.5 and 2 mM). Normal culture medium (isotonic, 300 mOsm/l) served as the control for the experiment. After exposure for desired time periods, the monolayer cultures were washed with D-Hanks and the cells were removed by 0.25% trypsin.

Intracellular free Na⁺ concentration ([Na⁺]) was detected using the dye CoroNa™ Green (cat. no. C36676; Invitrogen; excitation 488 nm, emission at 516 nm) as previously described (16). Briefly, the H9c2 cells were cultured in six-well plates and treated with a NaCl concentration gradient (final concentrations were 25, 50, 100 and 150 mmol/l) for 24 h; after adding CoroNa Green (5 µmol/l), the fluorescence signal was detected by flow cytometry (Cytomic FC500; Beckman-Coulter, Inc.) and analyzed by FlowJo software (version 7.6.1; TreeStar Inc.).

ROS production was detected using the dye 2,7-dichlorofluorescein diacetate (DCFH-DA; MilliporeSigma). The H9c2 cells were pretreated with or without ROS scavenger NAC (0.5, 2 mM) for 1 h followed by incubation with NaCl (50 mM), mannitol (100 mM) or control medium for 90 min. Cells were removed by 0.25% trypsin with culture medium without FBS. DCFH-DA (10 µmol/l) was added to the H9c2 cells and incubated at 37˚C for 30 min in dark. Analysis of apoptosis was performed by an Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) staining kit (BioLegend, Inc.). Briefly, H9c2 cells were seeded onto six-well plates and incubated with increased medium mannitol (100 mM), NaCl (50 mM) and NaCl (50 mM) supplemented with ROS scavenger NAC (0.5, 2 mM) for 24 h. Thereafter, the cells were harvested and washed twice with D-Hanks, centrifuged (4˚C, 350 x g, 5 min) and added 1X binding buffer (500 µl), Annexin V-FITC (2.5 µl) and PI (5 µl), incubated at room temperature in dark for 15 min then filtrated and kept on ice for an immediate detection by a FC500 flow cytometer.

H9c2 cells were washed and fixed with 4% paraformaldehyde at 20˚C for 15 min. Then the cells were blocked in fetal bovine serum for 20 min and stained with primary antibodies against LC3 (1:500; cat. no. NB100-2220; Novus Biologicals, LLC) for 2 h at 37˚C. After washing four times with PBS for 5 min, cells were incubated with rhodamine red-conjugated secondary antibodies (1:100; cat. no. CW0161; Beijing Kangwei Century Biotechnology Co., Ltd.) for 1 h at 37˚C and the nuclei were counterstained with DAPI (1:100, Vector Laboratories, Inc.) for 20 min. Then images were examined with a fluorescent microscope (80i; Nikon Corporation). The median fluorescence intensity of LC3 positive area was determined by Image Pro Plus software (version 4.5; Media Cybernetics, Inc.).

Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Briefly, the H9c2 cells were washed twice with D-Hanks and lysed with 1 ml of TRIzol^®. Subsequently, total RNA was extracted by adding chloroform and subsequent centrifugation (4˚C, 12,000 x g, 5 min) and precipitation with isopropyl alcohol. The RNA concentration and purity were measured by NanoDrop 2000C (Thermo Fisher Scientific, Inc.) and the ration of optical density value (260/280 nm) was at 1.8 to 2.0. Total RNA (2 µg) was reverse-transcribed into cDNA using a reverse transcription kit (Promega Corporation) in 25-µl reaction volumes according to the manufacturer's instructions. Transcript expression levels were quantified by SYBR Green RT-qPCR using an ABI PRISM 7300 sequence detector system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used for PCR analysis are shown in Table I. The conditions for amplification were as follows: 95˚C for 10 min, followed by 40 cycles at 95˚C for 15 sec and 60˚C for 60 sec. The relative expression levels were calculated using the 2^-ΔΔCq method (17).

For in vivo experiments, the heart tissue was homogenized in the RIPA lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, with PMSF (100:1), incubated on ice for 30 min to obtain the whole-cell protein extraction, followed by centrifugation at 4˚C and 12,000 x g for 10 min. For in vitro examinations, the H9c2 cells were washed and centrifuged then lysed in RIPA buffer for 30 min on ice to obtain the protein extraction. The protein concentration was determined with BCA protein assay kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions with bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) as the standard. For western blotting, equal amounts of protein samples (50 µg) were added denaturing buffer and subjected to a 10 min boil at 99.5˚C. Following SDS-PAGE on 10 or 15% polyacrylamide gels, proteins were transferred onto PVDF membranes using the Trans-Blot Turbo blotting system (Bio-Rad Laboratories, Inc.) and incubated in 5% skim milk in PBST (PBS containing 0.1% v/v Tween 20) for 2 h at 37˚C, then respectively probed with primary antibodies against lysosome-associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc-17768; Santa Cruz Biotechnology, Inc.), light chain 3LC3; 1:1,000; cat. no. NB100-2220), autophagy-related gene (ATG) 9 (1:1,000; cat. no. NB110-56893), ATG5 (1:500; cat. no. NB110-53818), Beclin1 (1:8,000; cat. no. NB110-87318), ATG16L1 (1:1,000; cat. no. NB110-60928; all from Novus Biologicals, LLC), GAPDH (1:2,000; cat. no. ab181602; Abcam) at 4˚C overnight. After washing four times with PBST for 15 min, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, cat. no. 6721, 1:2,000; goat anti-mouse IgG, cat. no. 6789, 1:2,000; Abcam) for 40 min at 37˚C. After washing fourth with PBST for 15 min, protein signals were detected by enhanced chemiluminescence method. The protein signal was amplified with Western Chemiluminescent Horseradish Peroxidase Substrate (MilliporeSigma) and detected using a Chemi DOC XRS+ Imaging System (Bio-Rad Laboratories, Inc.). Densitometric analyses were performed with Image J software (version 1.41o; National Institutes of Health).

All data are presented as the mean ± SEM. Statistical analysis was determined by independent sample unpaired t-test or one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test using the GraphPad Prism v5 (GraphPad Prism Software Inc.). Two-tailed P<0.05 was considered to indicate a statistically significant difference.

Body mass was comparable among all of the groups after 8 weeks of dietary intervention (Fig. 1). Throughout the 16 weeks of dietary intervention, there was a significant decrease in body weight in the SHR fed with the HS diet, which indicates a deteriorating health status. Moreover, the HS diet also led to an increase in LV mass, cardiomyocyte hypertrophy and collagen deposition in SHR and WKY compared with their LS-diet-fed counterparts (Figs. 1 and 2). Invasive LV hemodynamic analysis revealed that the HS diet progressively impaired the LV systolic (SBP and +dp/dt_max) and diastolic (LVEDP and -dp/dt_min) functions of the SHR in a time-dependent manner (Fig. 1). These results demonstrated that a transition from compensated LV hypertrophy to decompensation occurred when the HS diet intervention persisted for 12 weeks in the SHR. A time-dependent increase in collagen deposition and LVEDP was also noted in the LS-fed SHR and HS-fed WKY rats, but an obvious deterioration of the LV systolic and diastolic functions was not observed during this period in these two groups (Figs. 1 and 2).

After 12 weeks of dietary intervention, massive vacuole-like structures were observed in LV tissue from HS-diet-fed SHR, compared with LS-diet-fed SHR (Fig. 3A and B). To examine the potential reasons accounting for these structures, various staining methods and TEM were performed for pathological examinations (Fig. 3). The results showed that these cardiac vacuoles observed in the HS-fed SHR under light microscopy are due to autophagic vacuolization (double-membrane vesicles containing cytoplasmic organelles). Depending on the engulfed structures (from protein aggregates, intracellular organelles to pathogens), the size of the autophagosomes can range from 0.5 to 10 µm in diameter (18). As revealed in Fig. 3, the size of these vacuole-like structures, observed under light microscopy, could be even larger than 20 µm in diameter; this finding has previously been reported in humans (19), and may be due to the fusion of the autophagosomes.

Next, autophagy-associated key proteins in LV tissue from SHR sacrificed at different time points were examined by western blotting (Fig. 4). The results demonstrated a global change of the autophagy since SHR was fed by HS diet for 12 weeks. This change persisted to 16 weeks in SHR on the HS diet, as shown by upregulated Beclin 1/ATG6 (required for autophagy induction and nucleation), ATG16-ATG5-ATG12 and LC3-II (two complexes required for phagophore formation and autophagosome expansion), LAMP1 (maker for lysosome fusion with the autophagosome to form an autolysosome) and ATG9 (necessary for most ATG protein recycle). These results clearly demonstrated a temporal and spatial association between autophagic change and LV function deterioration in the HS-fed SHR. Similarly, but to a lesser extent (not global), autophagic change was observed in the HS-fed WKY (Fig. 5).

Increasing evidence shows that chronic HS intake could induce Na⁺ accumulation in the interstitial space without a commensurate retention of water, which yields increased tonicity (18,20,21). As shown in Fig. 6, upregulation of myocardial TonEBP was also demonstrated in the HS-fed WKY and SHR. In addition, using LV ashing samples, LV tissue composition analysis of Na⁺, K⁺ and water was performed in SHR after dietary intervention for 8 weeks. The results revealed that cardiac extracellular [Na⁺] was markedly higher than plasma level (215.0±2.0 mmol/l vs. 139.1±1.84 mmol/l) in LS-diet-fed SHR, and this difference was further increased in HS-diet-fed SHR (237.0±6.0 mmol/l, Table II). To examine the impact of increased extracellular [Na⁺] on the autophagic response in rat H9c2 cardiomyocytes, an additional NaCl concentration gradient was added in the culture medium. It was identified that there was a proportional increase of cytosolic [Na⁺] as extracellular [Na⁺] increased (Fig. 7A), indicating a Na⁺ leak into the cytosol when the cells were exposed to high [Na⁺]. A previous study showed that HS loading with a diet containing 8.0% NaCl in Sprague-Dawley rats for 2 weeks led to an increase of [Na⁺] ~40 mM in the skin compared with serum [Na⁺] (15). Based on the relationship between extracellular and intracellular [Na⁺] in H9c2 cells and cardiac extracellular [Na⁺] (Fig. 7A and Table II), an additional 50 mM NaCl was thus used in the culture medium, which equals 400 mOsm/l (serum osmolality is ~300 mOsm/l), to simulate the extracellular hyperosmolarity induced by chronic HS intake in the subsequent in vitro studies. It was found that incubation with an additional 50 mM NaCl induced an upregulation of key components of autophagy at both the mRNA and protein levels (Fig. 7B and C).

In addition, it was identified that the exposure of H9c2 cells to an additional 50 mM NaCl in the culture medium led to an enhanced intracellular ROS production (Fig. 8A), as well as increased pro-apoptotic response (Fig. 8B). When NAC was used as a ROS scavenger, the high NaCl-induced upregulation of pro-apoptotic response was significantly attenuated (Fig. 8B). Similarly, when exposure to the high extracellular NaCl occurred, there was an upregulation of key autophagy components both at mRNA and protein levels, as well as increased autophagosome formation (Fig. 9), which could be suppressed by NAC, implying that high [Na⁺]-induced autophagic change is ROS-dependent. Notably, when 100 mM mannitol (400 mOsm/l) was used as a positive control at the same extracellular hyperosmolality with 50 mM NaCl, the magnitude of ROS generation (Fig. 8) and the pattern of key autophagic component upregulation were different from those achieved by 50 mM NaCl, providing evidence to indicate that high [Na⁺]-induced changes in H9c2 cells are not entirely dependent on extracellular osmolality (Fig. 9A-D).