The a-MHC-cTnT^R141W transgenic mice were generated in the Key Laboratory of Human Disease Comparative Medicine and maintained in a C57BL/6J genetic background in the Key Laboratory of Human Disease Comparative Medicine (Ministry of Health, Peking Union Medical College, Beijing, China), and exhibited DCM phenotypic characteristics consistent with those reported previously (4). A total of 58 4-month-old mice were used, including 40 cTnT^R141W transgenic mice and 18 wild-type mice. In total, there were 34 males weighing 28–32 g, and 26 females weighing 22–24 g. All mice were bred in an American Association for Accreditation of Laboratory Animal Care-accredited facility, housed at 21±2°C with 50±5% relative humidity under a 12-h day/night cycle and had free access to food and drinking water. The procedures were approved by the Animal Care and Use Committee at the Institute of Laboratory Animal Science, Peking Union Medical College (approval no. ZLF18004).

The 4-month-old male and female cTnT^R141W transgenic mice were randomly assigned to treatment groups. DAS (cat. no. A35801; Sigma-Aldrich; Merck KGaA) was diluted in corn oil to a final concentration of 80 mg/ml. In the treatment groups, cTnT^R141W transgenic mice were administered DAS at a dose of 200 (n=12) or 400 mg/kg (n=10) via intraperitoneal injection three times weekly for 6 weeks. A group of cTnT^R141W transgenic mice (n=9) and non-transgenic littermates (NTG; n=8) were treated with corn oil (0.1 ml per mouse) as the placebo control and wild-type normal control, respectively. As a commercially available drug control, a group of cTnT^R141W transgenic mice were treated with enalaprilat (cat. no. H20010498; Changzhou Pharmaceutical Factory) via intraperitoneal injection three times weekly for 6 weeks, an angiotensin-converting enzyme (ACE) inhibitor that has been widely used in the clinical treatment of DCM and HF (33,34), at a dose of 0.76 mg/kg (n=9). The dose of DAS was selected based on previous reports (35,36). Therefore, there were total five groups in this study, including NTG normal control, placebo model control, DAS treatment at high dose (400 mg/kg), DAS treatment at low dose (200 mg/kg) and enalaprilat. After the last echocardiograph examination, the animals were euthanasia via cervical dislocation.

Heart function and structure were analyzed via echocardiography once every 2 weeks during treatment. Briefly, the mice were lightly anesthetized via an intraperitoneal injection of 216 mg/kg tribromoethanol and then subjected to 2-D guided M-mode echocardiography with a 30-MHz transducer for echocardiographic examination (Vevo770; FUJIFILM VisualSonics Inc.). Measurements of left ventricular (LV) fractional shortening (LVFS), LV ejection fraction (LVEF), LV diameter at end systole (LVESD), LV diameter at end diastole (LVEDD), LV posterior wall at end systole (LVPWS), LV posterior wall at end diastole (LVPWD), LV anterior wall at end systole (LVAWS) and LV anterior wall at end diastole (LVAWD) were based on the analysis of ≥10 separate cardiac cycles.

For light microscopy, heart tissues were first fixed in 4% formaldehyde at room temperature for 24 h, embedded in paraffin and cut into 4-µm thick sections (4). Heart tissue sections were imaged after H&E and Masson staining. For H&E staining, the sections were stained using a Hematoxylin and Eosin Staining kit (cat. no. G1121; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, paraffin-embedded sections were dewaxed in xylene two times for 10 min each time at room temperature. The sections were rehydrated in a series of ethanol (100, 95, 85 and 75%) for 3 min per gradient, and for 2 min in distilled water. Subsequently, the sections were stained with hematoxylin stain for 3 min at room temperature and washed with distilled water to remove floating colors. The differentiation solution was differentiated for 3 min and washed with distilled water twice, for 2 min each time. The sections were put into eosin dye solution for 2 min, then washed with distilled water for 2–3 sec at room temperature. Then, the sections were proceeded to the dehydrated step through soaking in a series of concentrations of alcohol at room temperature (for 2–3 sec at 75, 85 and 95%, for 1 min at 100%). The sections were then incubated with xylene twice at room temperature (for 1 min each time), followed by sealing in neutral gum and finally observed under the microscope.

For Masson trichrome staining, the sections were incubated in celestine blue solution (cat. no. G1345; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min and briefly washed with H₂O. Then, sections were incubated in hemalun solution for 5 min and in fuchsine acid/ponceau xylidine (0.5% fuchsine acid, 1.5% ponceau xylidine, 1,75% glacial acetic acid) for 5 min. Subsequent, sections were briefly washed with H₂O and incubated in phosphomolybdic acid (1%) for 10 min and in aniline blue solution (2.5% anilin blue, 2.5% glacial acetic acid) for 5 min. Next, they were briefly washed with H₂O and incubated in acetic acid (1%) for 1 min. Finally, the sections were briefly incubated in an ascending ethanol series followed by xylol, before they were embedded.

Heart tissues (~2 mm³) from the free wall of the left ventricle were fixed in 2.5% glutaraldehyde (preparation for 100 ml solution: 50 ml of 0.2 M phosphate buffer, 10 ml of 25% glutaraldehyde and 40 ml double distilled water) at 4°C for 12 h (4). After washing three times with phosphate solution (pH=7.2), the samples were fixed in 1% osmium acid at 4°C for 2 h. Dehydration was carried out sequentially with four graded concentrations of ethanol between 50–100% at room temperature. After epichlorohydrin replacement and epoxy embedding at room temperature, tissue sections were cut on an ultramicrotome. The semi-thin sections (thickness, 900 nm) were further stained with an equal mixture of 4% uranyl acetate and acetone for 30 sec at room temperature, and then in lead citrate for 2 min. The final ultra-thin sections (90 nm) were analyzed under a JEM-1230 transmission electron microscope (JEOL Ltd.).

After treatment, the mice were sacrificed and total lysates were prepared as previously reported (4). H₂O₂, malondialdehyde (MDA; lipid peroxidation) and glutathione (GSH) in heart tissues were measured with relevant assay kits (H₂O₂ Assay kit, cat. no. ab102500; MDA Assay kit, cat. no. ab118970; GSH/GSSG Ratio Detection Assay kit, cat. no. ab138881; all from Abcam) following the manufacturer's procedures.

For the H₂O₂ assay, 10 mg tissue was first washed in cold PBS and homogenized in 500 µl assay buffer with a Dounce homogenizer sitting on ice. The sample was centrifuged 13,000 × g for 5 min at 4°C at top speed using a microcentrifuge to remove any insoluble material. Finally, the content of H₂O₂ in the heart tissue was measured with a microplate reader at excitation/emission (Ex/Em)=535/587 nm.

For the lipid peroxidation assay, 10 mg tissue was first washed in cold PBS and homogenized in 303 µl lysis solution with a Dounce homogenizer sitting on ice. The sample was centrifuged at 13,000 × g for 10 min at 4°C to remove insoluble material. Then, thiobarbituric acid (TBA) reagent was added into the supernatant to generate MDA-TBA adduct. Finally, the absorbance was measured immediately at Ex/Em=532/553 nm.

For the glutathione assay, 10 mg tissue was first washed in cold PBS, then was resuspended in 400 µl cold lysis buffer. The samples were homogenized and centrifuged at 13,000 × g for 15 min at 4°C to remove any insoluble material. Next, 50 µl GSH assay mixture was added into each GSH standard or collected sample supernatant. Finally, the absorbance was measured at Ex/Em=490/520 nm with a fluorescence microplate reader.

For apoptotic cell staining, paraffin sections of heart tissues were stained using an ApopTag Plus Peroxidase In Situ Apoptosis kit (cat. no. S7101; MilliporeSigma) following the manufacturer's procedures. In briefly, heart tissues were first fixed in 4% formaldehyde at room temperature for 24 h, embedded in paraffin and cut into 4-µm thick sections. The tissue slides were rehydrated and were treated with 25 µg/ml proteinase K at 37°C for 8 min at room temperature. After washing and incubation with equilibration buffer for 5 min at room temperature, Tdt was diluted at 1:3.9 with reaction buffer and added to heart sections for 1 h at 37°C. After applying stop solution for 10 min at room temperature, the samples were incubated with anti-digoxigenin peroxidase conjugate at 37°C for 30 min. Slides were first treated with a 1:20 dilution of diaminobenzidine (3,3′-diaminobenzidine) substrate for 1 min at room temperature, then counterstained with hematoxylin (cat. no. G1121; Beijing Solarbio Science & Technology Co., Ltd.) for 30 sec at room temperature. The sections were proceeded to the dehydrated step through soaking in a series of concentrations of alcohol at room temperature (for 2–3 sec at 75, 85 and 95%, for 1 min at 100%). Then, the sections were incubated with xylene twice at room temperature (for 1 min each time), followed by sealing in neutral gum and finally observed under the microscope. In total, nine visual fields were randomly selected in each group to observe the apoptosis under a light microscope (BX53; Olympus Corporation; magnification, ×400). The results are expressed as the percentage of apoptotic cells among the total cell population.

Total RNA was isolated from heart tissues using TRIzol^® reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) and were used to synthesize cDNA with a RT kit (cat. no. RR820A; Takara Bio, Inc.) following the manufacturer's procedures. mRNA expression levels of procollagen type III α1 (Col3α1) was detected via RT-PCR using GAPDH for normalization under standard conditions. The primers were as follows: Col3α1 forward, 5′-CTCAAGAGCGGAGAATACTGG-3′ and reverse, 5′-CAATGTCATAGGGTGCGATA-3′; and GAPDH forward, 5′-CAAGGTCATCCATGACAACTTTG-3′ and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.

Heart tissues were homogenized and extracted using lysis buffer [tissue protein extraction reagent (cat. no. 78510); protease inhibitor cocktail (cat. no. 87785); phosphatase inhibitor cocktail (cat. no. 78420); PMSF (100 µM; cat. no. 36978; all from Thermo Fisher Scientific, Inc.); 100:1:1:1 ratio for configuration], followed by fractionation using a Mitochondrial/Cytosol Fractionation kit (cat. no. ab65320; Abcam) to obtain the cytosolic and mitochondrial fractions. An enhanced BCA protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology) was used to quantify the protein concentration. The total lysates, cytosolic and mitochondrial fractions (50 µg) were separated via 15% SDS-PAGE and transferred to a nitrocellulose membrane (Immobilon NC; MilliporeSigma). Following blocking in 5% fat-free milk in TBS-0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies targeted against: CYP2E1 (1:500; cat. no. ab28146), cardiac troponin T (1:500; cTnT; cat. no. ab8295), cytochrome c (1:500; cat. no. ab13575), procaspase 3 (1:1,000; cat. no. ab13847), procaspase 9 (1:1,000; cat. no. ab47537; all from Abcam), cleaved (active) caspase 3 (1:500; cat. no. 9507S) and cleaved caspase 9 (1:500; cat. no. 9664S; both from Cell Signaling Technology, Inc.), anti-β-tubulin (1:1,000; cat. no. ab21058) and anti-voltage dependent anion channel 1 (1:1,000; cat. no. ab14734; both from Abcam) were used. Following washing with TBST, the membranes were incubated with a HRP-conjugated secondary antibody (1:10,000; anti-rabbit IgG, cat. no. ZB-2301 or anti-mouse IgG, cat. no. ZB-2305; ZSGB-BIO, Inc.) at room temperature for 1 h. Primary antibody binding was visualized using a chemiluminescent detection system (Western Blotting Luminal Reagent; Santa Cruz Biotechnology, Inc.) and analyzed using the densitometry function of Quantity One software (version 3.0; Bio-Rad Laboratories, Inc.).

H9c2 cells (National Laboratory Cell Resource Sharing Service Platform) were cultured in DMEM (Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Cells were treated with DAS (12 h; 100 µmol/l; cat. no. A35801; Sigma-Aldrich; Merck KGaA) or isoprenaline hydrochloride (ISO; 12 h; 50 µmol/l; cat. no. I5627; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂ as required.

Cell viability was evaluated using calcein-AM/PI double staining following the manufacturer's instruction (cat. no. C542; Dojindo Laboratories, Inc.). Briefly, H9c2 cells were seeded in 96-well plates in triplicate at a density of 1×10⁴ cells/100 µl/well with 100 µl culture medium and cultured for 24 h. The cells were treated with ISO (50 µmol/l) or DAS (100 µmol/l) for 12 h at 37°C with 5% CO₂ and divided into four groups, including control, ISO, control + DAS and ISO + DAS groups. The cells were then washed twice with PBS and the working solution (PBS: calcein-AM: PI=1,000:1:1) and incubated for 15 min at 37°C. Live cells with yellow-green fluorescence and dead cells with red fluorescence (Leica Microsystems GmbH) were observed using a fluorescence microscope (magnification, ×100) at 490±10 nm and 545 nm excitation wavelengths. In total, three images of each well were captured for a total of three replicate wells for counting.

All experiments were performed ≥3 times. Statistical analyses were performed using SPSS software (version 19.0; IBM Corp.). The data were analyzed using two-tailed unpaired t-tests and one-way ANOVA followed by Tukey's post hoc analysis. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Compared with the NTG group, the cTnT^R141W transgenic mice showed typical DCM phenotypes, such as dilated chambers, thin walls and cardiac dysfunction. The DCM phenotypes were evidenced by increases in LVESD and LVEDD, and decreases in LVPWS, LVPWD, LVAWS, LVAWD, LVEF and LVFS (Fig. 1A-I; Table SI,Table SII,Table SIII).

Firstly, after 2 weeks of treatment, the early stage of drug intervention, it was found that improvement was already observed in both DAS groups, and echocardiographic parameters in both DAS groups showed an improved inhibition trend compared with that of the enalaprilat group. For example, LVESD decreased by 26.5 and 25.9% in the DAS high and low dose group, respectively, compared with the placebo model control group (P<0.001; Fig. 1B), while LVESD decreased by 18.9% in the enalaprilat group compared with the placebo model control group (P<0.01; Fig. 1B). Moreover, LVEF increased by 57.8 and 43.1% in the DAS high and low dose group, respectively, compared with the placebo model control group (P<0.001; Fig. 1H), while LVEF increased by 35.2% in the enalaprilat group compared with the placebo model control group (P<0.05; Fig. 1H).

After 6 weeks of treatment, LVESD decreased by 22.4% in the DAS high dose group (P<0.001; Fig. 1B), while it decreased by 11.1% in the enalaprilat group (P<0.05; Fig. 1B) compared with the placebo model control group. There was a significant increase in LVEF in the DAS high dose group compared with the placebo model control group (P<0.001; Fig. 1H), while there was no difference between the enalaprilat group and the placebo model control group (P>0.05; Fig. 1H). Furthermore, there was a significant increase in LVAWD in the enalaprilat group compared with the DAS high dose group (P<0.001; Fig. 1G). LVPWS increased in both the DAS high dose group and the enalaprilat group compared with the placebo model group (P<0.05; Fig. 1D), while it showed no significant difference between these two groups (P>0.05).

In summary, both DAS groups and the enalaprilat group exhibited a significant improvement on DCM phenotypes, as evidenced by changes in cardiac function, chamber size and wall thickness. While each factor has its advantages, DAS exhibited a stronger effect on control of chamber dilation and dysfunction, and the enalaprilat group was more effective at increasing wall thickness. Furthermore, this improvement on morphology and function of model mice occurred earlier in both DAS groups compared with that of the enalaprilat group.

After drug treatment, hearts from all five groups were sampled for gross morphology and pathological examinations.

First of all, the protein expression level of CYP2E1 was determined via western blotting after treatment with different doses of DAS. Compared with the placebo model control group, the protein expression level of CYP2E1 in 200 and 400 mg/kg DAS groups decreased by 35.5% (n=3; P<0.01) and 51.8% (n=3; P<0.001; Fig. 2A and B), respectively. Moreover, CYP2E1 protein expression decreased by 35.9% in the enalaprilat group (n=3; P<0.01; Fig. 2A and B). The expression level of cTnT protein was also analyzed. The mutant form of cTnT is the human mutant form that was introduced by the transgenic method, which is the cTnT band at the higher molecular weight in Fig. 2A, and the location of endogenous cTnT proteins in mouse myocardium was slightly lower than that of exogenous mutants. The expression level of the cTnT showed no difference between the three therapeutic groups and the placebo model group (Fig. 2A and C).

The increased heart to body weight ratio of cTnT^R141W mice was reversed by DAS treatment to almost the normal level both in the 400 and 200 mg/kg group (P<0.05; Fig. 2D). DAS treatment significantly improved the chamber dilation, wall thinning and myocyte disarray in cTnT^R141W DCM model mice, as determined via H&E staining (Fig. 2E and F). Through ultrastructure observation using TEM, it was found that DAS treatment improved the poor myofibril organization, including diffusion, damage and lysis, in cTnT^R141W mice (Fig. 2H). Quantitative analysis of the Masson staining and the mRNA expression level of Col3α1 showed that collagen deposition in the interstitial space of cTnT^R141W mice was significantly reduced in both DAS and enalaprilat groups compared with the placebo model control group (Fig. 2G and I-K).

Both DAS and enalaprilat treatment exhibited a favorable therapeutic effect on improving of the destroyed microstructure and ultrastructure of the myocardium in DCM model mice. Moreover, the DAS group exhibited a greater advantage in reducing index of heart to body weight ratio.

To verify the ameliorative effect of DAS on the pathological phenotype of cardiomyopathy in vitro. Cell experiments were performed, and it was found that DAS could significantly inhibit the increased expression of CYP2E1 induced by ISO treatment in H9c2 cells, as well as significantly improve the cell death induced by ISO (Fig. S1), which were all consistent with the results in vivo.

CYP2E1 catalyzes the production of ROS even in the absence of substrate, leading to oxidative stress (37). Therefore, H₂O₂, MDA and GSH levels were measured as indicators of oxidative stress in all five groups.

H₂O₂ and MDA levels were significantly decreased (P<0.001; Fig. 3A and B), while GSH levels were significantly increased (P<0.01 and P<0.001; Fig. 3C) in both 400 and 200 mg/kg DAS treatment groups compared with those of the placebo model group. Inhibition of the expression of CYP2E1 by DAS resulted in a reversion of the levels of H₂O₂, MDA and GSH in the myocardium to almost the normal levels in both the 400 and 200 mg/kg DAS groups. By contrast, no significant differences in those three indicators were observed in the enalaprilat group compared with the placebo model group (P<0.05; Fig. 3A-C).

Therefore, inhibition of oxidative stress level is one of the possible underlying mechanisms of DAS, rather than enalaprilat, that is involved in the protection against the pathological process of DCM.

The induction of CYP2E1 causes cytochrome c release and activation of the mitochondrial apoptosis pathway in the heart in cTnT^R141W DCM model mice (4).

In both DAS groups, the increased release of cytochrome c in cTnT^R141W DCM model mice almost reached those of normal levels (P<0.05; Fig. 4A and B). The activation of caspase 9 was decreased by 66.6% (P<0.001) and 66.9% (P<0.001; Fig. 4A and C) in the 400 and 200 mg/kg DAS groups, respectively, compared with the placebo model group. The activation of caspase 3 was decreased by 65.1% (P<0.001) and 62.7% (P<0.001; Fig. 4A and D) in the 400 and 200 mg/kg DAS groups, respectively, compared with the placebo model group.

The release of cytochrome c from the mitochondria to the cytoplasm triggers the apoptosis of cardiac myocytes in cTnT^R141W model mice (38,39). It was determined that there were 69.3 and 64.7% decreases in proportion of apoptotic cells in the 400 (P<0.05) and 200 mg/kg groups (P<0.05; Fig. 4E and F), respectively, compared with the placebo model group. Furthermore, DAS and enalaprilat exhibited no difference in apoptosis inhibition.

Therefore, inhibition of the mitochondrial apoptosis pathway is one of the possible mechanisms of DAS that is involved in the protection against the pathological process of DCM.