A total of 8 male heart/muscle-specific Mn-SOD-deficient (H/M-Sod2^−/−) mice (age, 8 weeks; weight, 22.5±3.1 g) were purchased from The Fourth Affiliated Hospital of Harbin Medical University (Harbin, China). The mice were kept in a temperature-(20–24°C) and humidity-controlled (45–55%) environment, under a 12-h light/dark cycle, with free access to food and water. H/M-Sod2^−/− mice were fed with 3 mg/kg/day n-3 PUFA (n=4) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or vehicle (n=4) (lauric acid, Sigma-Aldrich; Merck KGaA) (18) for 10 weeks, and cardiac function was evaluated by echocardiography. Mice were subsequently sacrificed, hearts were harvested and the proteins extracted. The present study was approved by the Zhejiang Province Hospital of Integrated Traditional Chinese and Western Medicine (Hangzhou, China).

Two-dimensional (2D) echocardiography was performed with certain modifications according to previously reported methods (19). On the day of evaluation, sodium pentobarbital (50 mg/kg; P3761, Sigma-Aldrich; Merck KGaA) was administered by intraperitoneal injection to anesthetize the mice. After shaving the chest, 2D echocardiography was conducted with an echocardiographic system (model SSD-900; Hitachi-Aloka Medical, Ltd., Tokyo, Japan) and 7.5 MHz probe (UST-987-7.5; Hitachi-Aloka Medical, Ltd.). Furthermore, the fraction shortening (FS), ejection fraction (EF), left ventricular internal diameter (LVID) during systole and diastole, and endsystolic and end-diastolic volumes were calculated using Vevo Vasc Analysis software (version 2.2.3; Fujifilm VisualSonics, Inc., Toronto, Canada) as previously described (20).

Primary cardiomyocytes from mouse neonatal hearts were isolated as previously described (21). In brief, hearts were isolated and digested with collagenase type II solution (Worthington Biochemical Corporation, Lakewood, NJ, USA). Following digestion, the cells were plated in a 25-cm² culture flask for 2 h at 37°C in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to collect cardiomyocytes. Attached cells were considered as non-myocytes and discarded, and the unattached cells were primarily cardiomyocytes.

For the histological analysis, heart tissues were fixed at room temperature for 10 min in 10% buffered formalin. Fixed tissues were subsequently dehydrated, embedded in paraffin, sectioned into 4-µm slices and stained with hematoxylin and eosin (H&E). Furthermore, Masson's trichrome was used to stain myocardial sections in order to evaluate the cardiomyocyte diameter and degree of fibrosis. Images were captured with a Pixera Pro600EX camera and a VANOX-S microscope (Olympus Corporation, Tokyo, Japan). Additionally, the fibrotic area and cardiomyocyte diameter (>30 cells) were quantified with Qwin Plus V3 (Leica Microsystems, Inc., Buffalo Grove, IL, USA), and the collagen volume percentage was calculated as the mean of 5 fields for each animal.

Primary cardiomyocytes were cultured in 6-well tissue culture plates (1×10⁵ cells per well). The cells were incubated at 37°C in serum-free DMEM for 16 h at 70–80% confluence. The cells were then washed three times with cold PBS and fixed with 4% formaldehyde (Zhongshan Technology Co., Ltd., Zhongshan, China) in PBS for 20 min at room temperature. Next, the cells were washed three times with cold PBS and stained with Hoechst 33258 (10 µg/ml; 50 µl/slide; Sigma-Aldrich; Merck KGaA) at room temperature for 5 min. Following staining, cold PBS was used to further rinse the cells, and were then examined under a fluorescence microscope.

In order to detect the effects of n-3 PUFA on cell apoptosis, primary cardiomyocytes isolated from mice were washed with cold PBS three times. Next, flow cytometry was used to determine cell apoptosis with an Annexin-V fluorescein isothiocyanate-propidium iodide (FITC-PI) apoptosis kit (Invitrogen, Carlsbad, CA, USA). In summary, the cells (1×10⁶) were washed with 1X PBS three times and suspended at 2–3×10⁶ cells/ml in X1 Annexin-V binding buffer [10 mM HEPES/NaOH, (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂]. Annexin-V FITC and PI buffer were then added to the cells, which were then incubated at room temperature for 15 min in the dark. The cells that did not undergo any treatment were used as an internal control. Following incubation, the cells were filtered using a filter screen and analyzed using a flow cytometer within 1 h of staining. Cell apoptosis was analyzed using BD CellQuest Pro^™ Analysis Software (BD Biosciences, San Jose, CA, USA).

Primary cardiomyocytes isolated from the mice were cultured on slides in a 6-well chamber at 60% confluence at 37°C. Two days later, the slides were washed with cold PBS three times. Additionally, the slides were treated with 5 µM dihydroethidium (DHE; Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) in serum-free DMEM F-12 medium (GE Healthcare Life Sciences) for 30 min at 37°C in darkness. Furthermore, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature. The slides were then washed with cold PBS three times and mounted. Finally, immunofluorescence images were captured by fluorescence microscopy, and in order to quantify the intracellular ROS, the relative fluorescence intensities were analyzed using flow cytometry in the primary cardiomyocytes. Briefly, 1×10⁶ cells were centrifuged (200 × g) for 10 min at room temperature and the supernatant was discarded. The cell pellet was resuspended in 1 ml of PBS at room temperature. A 2 mM solution of H₂DCFDA (Invitrogen; Thermo Fisher Scientific, Inc.) was freshly prepared in ethanol and 5 µl was added to the cell suspension (final concentration 10 µM) and incubated at 37°C for 20 min. Cells were centrifuged at 200 × g for 5 min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in 500 µl of PBS. Flow cytometric analysis was performed in duplicate using a flow cytometer and acquired data were analyzed using WinMDI v2.8 software (BD Biosciences).

Cellular proteins were extracted using RIPA buffer [50 mM Tris/HCl, (pH 7.4), 150 mM NaCl 1% (v/v) NP-40, 0.1% (w/v) SDS; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China] with 0.3% (v/v) protease inhibitor (Sigma-Aldrich; Merck KGaA), 1% (v/v) phenylmethane sulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich, Inc.; Merck KGaA) for 15 min at room temperature. The lysates were collected for the total protein following centrifugation at 10,000 × g at 4°C for 15 min. Furthermore, the bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 15 µg protein per lane was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking with 8% (w/v) milk in PBST for 2 h at room temperature, the membranes were then incubated with primary antibodies against GAPDH (cat. no. 2118; 1:5,000), cleaved-caspase 3 (cat. no. 9664; 1:1,000), p-JNK (cat. no. 4668; 1:1,000), JNK (cat. no. 9252; 1:1,000), p-NF-κB (cat. no. 3033; 1:1,000), NF-κB (cat. no. 8801; 1:1,000) (all Cell Signaling Technology, Inc., Danvers, MA, USA) and NOX4 (cat. no. ab109225; 1:1,000; Abcam, Cambridge, UK) overnight at 4°C. Next, the membranes were washed with TBST three times. Additionally, they were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or HRP-conjugated mouse anti-goat IgG (both 1:5,000; ZB-2301, Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 2 h at room temperature and then washed three times with TBST. Enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA) was used to determine the protein concentrations according to the manufacturer's recommendations. Finally, the relative contents of protein were normalized against GAPDH. All experiments were repeated three times. ImageJ 1.43b software (National Institutes of Health, Bethesda, MD, USA) was used for densitometry analysis.

The nuclear and mitochondrial fractions of the heart were isolated in order to quantify cardiac protein carbonylation. In brief, the crude nuclear fractions were isolated from tissue homogenates at 1,000 × g for 5 min at 4°C and washed with PBS at 4°C. The mitochondrial fractions were separated as previously described (22). The carbonylation of mitochondrial and nuclear protein was evaluated using an Oxyblot protein oxidation detection kit (EMD Millipore) according to the manufacturer's instructions. Immunoreactive spots were visualized with enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) and quantified with ImageJ 1.43b (National Institutes of Health, Bethesda, MD, USA). Furthermore, the ATP content was determined using an ATP assay kit (colorimetric/fluorometric) (cat. no. ab83355; Abcam), according to the manufacturer's instructions.

Data are presented as the mean ± standard error from three independent experiments. Statistical analysis was performed with Student's-test. P<0.05 was considered to indicate a statistically significant difference.

Compared with wild type (WT) mice, hearts from H/M-Sod2^−/− mice yielded increased parasternal long-axis M-mode echocardiographic images (Fig. 1A). As shown in Fig. 1B, H&E staining revealed an evident dilation of DCM hearts from H/M-Sod2^−/− mice compared with those from WT mice. Furthermore, the myocardial sections from H/M-Sod2^−/− mice indicated eccentric hypertrophy and myocardial disarray compared with hearts from WT mice (Fig. 1C). These data showed the cardiac dilation phenotype in hearts from H/M-Sod2^−/− mice.

To identify whether n-3 PUFA prevents cardiac enlargement in H/M-Sod2^−/− mice, Masson's trichrome staining demonstrated that interstitial fibrosis was markedly improved after n-3 PUFA treatment, suggesting that n-3 PUFA was able to partially reverse cardiac fibrosis in the H/M-Sod2^−/− mice (Fig. 2A). Furthermore, the cardiomyocyte diameter was significantly decreased in H/M-Sod2^−/− mice treated with n-3 PUFA, compared with untreated mice, indicating that cardiac hypertrophy was ameliorated by n-3 PUFA treatment (Fig. 2B). Following n-3 PUFA treatment, the percentage of FS was significantly enhanced, compared with untreated mice (Fig. 2C). These data suggested that n-3 PUFA treatment ameliorated cardiac dysfunction in H/M-Sod2^−/− mice.

The ROS production in primary cardiomyocytes isolated from H/M-Sod2^−/− and WT mice was identified. As shown in Fig. 3A, DHE staining revealed that n-3 PUFA treatment markedly decreased the ROS production in primary cardiomyocytes isolated from H/M-Sod2^−/−. Subsequently, oxidative protein damage was investigated in nuclei and mitochondria. Protein carbonylation was significantly decreased in the nuclei and mitochondria of primary cardiomyocytes of H/M-Sod2^−/− mice treated with n-3 PUFA (Fig. 3B and C). Furthermore, n-3 PUFA treatment recovered 21% more ATP content compared with the untreated H/M-Sod2^−/− mice (Fig. 3D). These data indicated that n-3 PUFA decreases ROS generation, protein damage and the ATP content in H/M-Sod2^−/− mice.

In order to investigate the potential role of n-3 PUFA on cell apoptosis, Hoechst staining and flow cytometry were applied to determine apoptosis of primary cardiomyocytes isolated from H/M-Sod2^−/− mice. Following n-3 PUFA treatment, primary cardiomyocytes isolated from H/M-Sod2^−/− mice after apoptosis were evidently decreased compared with the untreated group (Fig. 4A and B). Notably, the phosphorylation levels of c-Jun N-terminal kinases (JNK), nuclear factor (NF)-κB and cleaved-caspase 3 were significantly decreased with n-3 PUFA treatment, whereas the expression of NADPH oxidase 4 (NOX4) was significantly increased (Fig. 4C).