DAS Improves Cardiac Morphology Breakage and Dysfunction in cTnTR141W DCM Mice
The cTnTR141W transgenic mice displayed typical DCM phenotypes, with dilated chambers, thin walls, and cardiac dysfunction, which were successfully established in our previous study. In cTnTR141W DCM mice, CYP2E1 expression is induced in heart tissues, and knockdown of endogenous CYP2E1 level significantly prevents the development of DCM [4, 23].
To test the protective effect of DAS, an inhibitor of CYP2E1, on the development of DCM, we analysed cardiac morphology and function with M-mode echocardiography in the cTnTR141W DCM mice during treatment. And four-month-old cTnTR141W transgenic mice, typically develop DCM from this time of age, were randomly assigned to three treatment groups, including. DAS low-dose group (200 mg/kg, n = 12), DAS high-dose group (400 mg/kg, n = 10), positive drug control group (Enalaprilat, an angiotensin-converting enzyme (ACE) inhibitor that has been widely used in the clinical treatment of DCM and HF, 0.76 mg/kg, n = 9). The dose of DAS was selected based on other experimental studies [31, 32]. The dose of Enalaprilat was calculated from the ratio of mice to human weight. Non-transgenic littermates (NTG, n = 10) and a group of cTnTR141W transgenic mice (Placebo, n = 9) were treated with corn oil as the wild-type normal control and placebo control. Therefore, there are all five groups, including NTG, cTnTR141W, DAS treatment (400mg/kg), DAS treatment (200mg/kg), Enalaprilat treatment in this study.
The DAS treatment improved cardiac morphology breakage and dysfunction in a dose-dependent manner; furthermore, significant improvements were observed in the early stages of treatment compared with the Enalaprilat group (Fig. 1).
Compared with the non-transgenic group, the cTnTR141W transgenic mice showed typical FDCM phenotypes as dilated chambers, thin walls and cardiac dysfunction. It was evidenced by a 34.5% increase in LVESD (Fig. 1A- 1B and Table S1, n = 7, P < 0.001), a 19.7% decrease in LVPWS (Fig. 1A, 1C and Table S1, n = 7, P < 0.001), and a 37.7% decrease in LVEF (Fig. 1A, 1D and Table S1, n = 7, P < 0.001).
After 2 weeks of treatment, compared with the cTnTR141W group, left ventricle end-systole diameter (LVESD) decreased by 26.5% and 25.9% in the 400 mg/kg group (Fig. 1E and Table S1, n = 8, P < 0.001) and 200 mg/kg group (Fig. 1E and Table S1, n = 9, P < 0.001), respectively, but decreased by 18.9% in the Enalaprilat group (positive control, Fig. 1E and Table S1, n = 7, P < 0.01). Posterior wall at end systole (LVPWS) also increased significantly in both DAS groups (Fig. 1F and Table S1, n = 7, P < 0.01). There were 57.8% and 43.1% increases in left ventricle ejection fraction (LVEF) in the 200 mg/kg group (Fig. 1G and Table S1, n = 9, P < 0.001) and 400 mg/kg group (Fig. 1G and Table S1, n = 8, P < 0.001), compared with the cTnTR141W group, respectively, but only by 35.2% in the Enalaprilat group (positive control, Fig. 1G and Table S1, n = 7, P < 0.05).
After 6 weeks of processing, compared with the cTnTR141W group (placebo), LVESD decreased by 22.4% in the 400 mg/kg group (Fig. 1E and Table S3, n = 7, P < 0.001), while the Enalaprilat group (positive control) fell by only 11.1% (Fig. 1E and Table S3, n = 7, P < 0.05). LVPWS also increased significantly in the 400 mg/kg group compared with the placebo group (Fig. 1F and Table S3, n = 7, P < 0.05), but LVPWS between the 400 mg/kg group and the Enalaprilat group was no significant difference. There was a 50.2% increase in LVEF in the 400 mg/kg group (Fig. 1G and Table S3, n = 7, P < 0.001) compared with the cTnTR141W group, but did not increase significantly in the Enalaprilat group (Fig. 1G and Table S3, n = 7, P > 0.05), And all three parameters given no significance between the 200 mg/kg group and the placebo group.
Compared with Enalaprilat treatment, treatment with 400 mg/kg DAS resulted in a greater improvement of DCM dysfunction (Fig. 1H-1K), as evidenced by the changes in LVPWS and LVAWD (Fig. 1I and 1K). Furthermore, this improvement occurred earlier in both DAS groups than in the Enalaprilat group according to the significance analysis.
DAS Inhibits Cardiac Pathological Changes in cTnTR141W DCM Mice
After DAS treatment, hearts from all five groups were sampled for gross morphology examination and pathological examination.
First of all, we have analysed the expression of CYP2E1 protein by western blot, and which was dose dependent with the dose treated by DAS. The expression of CYP2E1 protein in 200 mg/kg and 400 mg/kg DAS groups decreased by 35.5% (Fig. 2A-2B, n = 3, P < 0.01) and 51.8% (Fig. 2A-2B, n = 3, P < 0.001). While the expression of CYP2E1 protein fell by 35.9% in the Enalaprilat group (Fig. 2A-2B, n = 3, P < 0.01). Furthermore, we have analysed the expression of cTnT protein, too. The mutant form of cTnT is the human mutant form which was introduced by the transgenic method, that 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 of the cTnT given no difference between three therapeutic groups and the model group (Fig. 2C)
The increased heart to body weight ratio of cTnTR141W mice was reversed by DAS treatment to nearly the normal level found in the 400 mg/kg group and 200 mg/kg group (Fig. 2A, n = 6, P < 0.05). DAS treatment significantly improved the chamber dilation, wall thinning, myocyte disarray in cTnTR141W mice, as analysed by hematoxylin-eosin (H&E) (Fig. 2E-2F). By contrast, Enalaprilat treatment failed to inhibit chamber dilation in cTnTR141W mice (Fig. 2D-2E, n = 6, P > 0.05). Through transmission electron microscopy (TEM) ultrastructure observation, DAS treatment also clearly improved the poor myofibril organization, including diffusion, damage and lysis, in cTnTR141W mice (Fig. 2H). Quantitative analysis of the Masson stain and the RT-PCR of col3α1 (Fig. 2I-2K) showed that collagen deposition in the interstitial space of cTnTR141W mice was reduced by DAS treatment (Fig. 2F-2G, n = 3, P < 0.001).
Although Enalaprilat treatment clearly improved most of the cardiac pathological changes, especially changes in microstructure and ultrastructure, DAS treatment exhibited superior effects in controlling dilation of the heart chamber in cTnTR141W mice.
DAS Reduces Oxidative Stress in cTnTR141W DCM Mice
CYP2E1 catalyses the production of ROS even in the absence of substrate, leading to oxidative stress. We measured hydrogen peroxide (H2O2), malondialdehyde (MDA) and glutathione (GSH) as indicators of oxidative stress levels in all five groups.
Compared with the placebo control group of cTnTR141W mice, H2O2 and MDA decreased (Fig. 3A-3B, n = 3, P < 0.001) while GSH augmented (Fig. 3A-3B, n = 3, P < 0.01) significantly in both 400 mg/kg and 200 mg/kg DAS treatment groups. By contrast, no significant differences in these three parameters were observed in the Enalaprilat group compared with the placebo control cTnTR141W group (Fig. 3A-3C, n = 3, P > 0.05).
Thus, inhibition of the expression of CYP2E1 by DAS resulted in reversion of the levels of H2O2, MDA and GSH in the heart to nearly the normal levels found in the 400 mg/kg group and 200 mg/kg group. However, Enalaprilat failed to control oxidative stress in the hearts of cTnTR141W DCM mice.
DAS Inhibits Mitochondrial Pathways of Apoptosis in cTnTR141W DCM Mice
The induction of CYP2E1 causes cytochrome c release and activation of the mitochondrial apoptosis pathway in the heart in cTnTR141W DCM mice [4]. In both DAS groups, the increased release of cytochrome c in cTnTR141W DCM mice was reversed to normal levels (Fig. 4A-4B, n = 3, P < 0.05). The activation of caspases 9 and 3 in the heart was inhibited by DAS treatment in cTnTR141W mice in a dose-dependent manner (Fig. 4A, 4C-4D). The animation of caspase 9 was fell by 66.6% (Fig. 4C, n = 3, P < 0.001) and 66.9% (Fig. 4C, n = 3, P < 0.001) in the 400 mg/kg and 200 mg/kg groups, respectively, compared with the placebo control cTnTR141W group. The animation of caspase 3 was fell by 65.1% (Fig. 4D, n = 3, P < 0.001) and 62.7% (Fig. 4D, n = 3, P < 0.001) in the 400 mg/kg and 200 mg/kg groups, respectively, compared with the placebo control cTnTR141W group.
The release of cytochrome c from the mitochondria to the cytoplasm triggers the apoptosis of cardiac myocytes in cTnTR141W mice. There were 69.3% and 64.7% decreases in apoptosis in the 400 mg/kg (Fig. 4E-4F, n = 3, P < 0.05) and 200 mg/kg groups (Fig. 4E-4F, n = 3, P < 0.05), respectively, compared with the placebo control cTnTR141W group (Fig. 4E-4F). Interestingly, Enalaprilat exhibited stronger effects than DAS on apoptosis inhibition, but DAS and Enalaprilat had equivalent inhibitory effects on the animation of the mitochondrial apoptosis pathway.