3-1. Physiological examinations
During the Dox treatment, body weight of animals dropped to a mean weight loss of 8.4
± 3.9% at week 4 and ultimately dropped to 11.4 ± 3.1% at week 12. In contrast, the sham control group gained a 20% increase in body weight during 12 weeks feeding and reached to a limit about 30 g. The significant differences (P < 0.01) between the groups and in comparison with the beginning were observed initially at week 2 and week 4, respectively (Figure 2).
Furthermore, Dox treatment significantly decreased the ventricle weight and the ventricle weight/tail length ratio beginning from week 3 and further declined in the process (Table 6).
The mortality of Dox-treated group was approximately 30% during 12 weeks. These mice died during week 5 to 12 primarily, even though Dox was not given to these mice during this time period.
3-2. Cardiotoxicity
Lead II electrocardiogram was introduced to monitor Dox-induced cardiotoxicity in this study according to the previous reports (van Acker et al., 1996). In contrast to ECG
recordings in humans, the ECG does not contain an S-T segment in mice. The T-wave immediately follows the QRS complex; thus, no S-T segment can be found (Figure 3B). The prolongation of the S-T interval in Dox-treated mice is secondary to an increase in action potential duration. The ST interval in the Dox-treated group increased from 24.2 ± 2.0 msec at week 1 to 35.0 ± 0.2 msec at week 12. The differences relative to sham control group were significant (P < 0.01) beginning from week 5 (Figure 3A).
Besides the electrophysiological tests, natriuretic peptides which were previously used as
biomarker of heart failure in several Dox-induced cardiomyopathy animal models were surveyed in this study (Rahman et al., 2001; Torrado et al., 2003). The mRNA levels for ANP and BNP from the LV tissue were increased 4.0 ± 1.8 fold (P < 0.01) and 1.6 ± 0.3 fold (P <
0.05) compared with control at week 12, respectively (Figure 4). The expression of GAPDH mRNA was used as internal control.
3-3. Histological examinations
The histological differences between control and Dox-treated mice were shown in Figure 5. Hearts were examined at week 12 with use of hematoxylin and eosin staining. The
ventricular sections demonstrated normal tissues in control mice (Figure 5A), whereas vacuolisation of cardiomyocytes existed in the Dox-treated mice (Figure 5B).
3-4. Expression of collagen type I and type III
We were interested in measuring the level of collagen type I and III in the tissue, which were predominant components of ECM. The transcript of collagen type I and III in the LV tissues were determined by semiquantitative RT-PCR (Figure 6A), and the expression of GAPDH mRNA was used as internal control. The expression level was presented as fold relative to control at each time point. After analysis, the statistical result showed no difference on mRNA expression level of collagen type I and III along the course (Figure 6B and 6C).
3-5. MMP-1: opposite trends of changes in latent and active form
Two major bands (57 kDa glycosylated latent proMMP-1 and 47 kDa active MMP-1) were detected on membrane by Western immunoblotting (Figure 7A). Comparing with
control, pro-MMP-1 in LV from Dox-treated mice increased 2.3 ± 0.7 fold (P < 0.01) at first week but trended down during the following stage. Except the first week, the amount of pro-MMP-1 in treated group was less than time-matched control mice with statistic
significance, even decreased about 5-fold in the end point (Figure 7B). In contrast, the active form of MMP-1 had a notable tendency of increase in the process. The active MMP-1
increased significantly 2.6 ± 0.6 fold (P < 0.01) initially at week 5 and further ascended to 4.7
± 0.7 fold (P < 0.01) at week 12 (Figure 7C). Collectively, an interesting pattern of the opposite trend of changes in latent and active form was illustrated in Figure 7D. Moreover, sum of these two forms was calculated as total MMP-1 protein roughly. Besides a decrease at week 3, there was no difference between two groups on total MMP-1 protein level (Figure 7E).
3-6. Decreased activity of MMP-2 and MMP-9
The LV tissue isolated from hearts with Dox-treated and control mice were used to detect the enzyme activity of two gelatinases, MMP-2 and MMP-9, by zymographic analysis (Figure 8A). As shown in Figure 8C, the 92 kDa pro-MMP-9 activity increased significantly by approximately 2 fold (P < 0.05) in the Dox-treated mice compared with control at first week but showed 2 fold decrease at week 12 (P < 0.01). The 68 kDa activation intermediate of MMP-2 displayed a similar pattern with pro-MMP-9 at the end stage; However, at week 12 MMP-2 only decrease about 1.5 fold (P < 0.05) (Figure 8B). In addition, a minor band on 72 kDa known as latent form MMP-2 showed no alteration in the whole process.
3-7. Decreased mRNA expression of MMP-2 and MMP-9
According to the results of zymographic assay, the down-regulated gelatinases activity
was observed in the LV of Dox–treated mice. Further, we measured the MMP-2 and MMP-9 mRNA levels by quantitative real-time PCR. The expression of GAPDH mRNA was used as internal control and the mRNA expression level were presented as fold related to control at each time point. The relative mRNA level of MMP-2 in the Dox group decreased 1.7 ± 0.2 fold and 1.6 ± 0.2 fold at week 9 and 12, respectively (Both P < 0.01) (Figure 9A). Similarly, the relative mRNA level of MMP-9 decreased 1.6 ± 0.2 fold and 3.8 ± 0.8 fold at week 9 and 12, respectively (Both P < 0.01) (Figure 9B). These reduced expression on gelatinases at the final stage and the greater changes in MMP-9 rather than MMP-2 corresponded with the results of gelatinases activity on zymogram.
3-8. Up-regulation of TIMPs activity
The proteolytic activity of MMPs can be regulated by TIMPs because of the inhibitive effect. Therefore, the relative abundances of TIMPs activity in the LV tissues were
determined by reverse zymography (Figure 10A). TIMPs inhibitory activity resulted in dark blue bands compared with typical SDS-PAGE. In which TIMP-2, -3 and -4 were identified upon molecule weight as 21, 24 and 22 kDa respectively. However, predicted 29 kDa TIMP-1 was undetectable. Generally, the TIMPs activity increased along the process. A significant 4.5
± 1.2 fold up-regulation of TIMP-2 was observed at week 12 (Figure 10B), the increase of TIMP-3 and TIMP-4 even earlier at week 5 (P < 0.05) (Figure 10C and 10D).
3-9. mRNA expression level of TIMPs
Comparing with the activity of TIMPs, the mRNAs of TIMPs in the LV tissues were also determined by semiquantitative RT-PCR (Figure 11A). In this analysis, the expression of GAPDH mRNA was used as internal control and the expression level were presented as fold
related to control at each time point. Except the 1.8 fold increase of TIMP-4 at week 9 and 12 (P < 0.05) (Figure 11E), there were no significant differences on mRNA expression of residual TIMPs in this study (Figure 11B-D).
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