Abstract
Background
It is not clear yet how tadalafil affects nonischemic cardiomyopathy, although its beneficial effects on acute myocardial infarction are well-known. We investigated tadalafil's beneficial effects on nonischemic cardiomyopathy and the specific mechanisms of its effects.
Methods
Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, tadalafil (4 mg/kg/day, p.o., 14 days) was started simultaneously. After two weeks, cardiac function was evaluated by echocardiography and cardiac catheterization, then all of the mice were killed and cardiac specimens were subjected for hemotoxylin and eosin staining, Masson's trichrome staining, terminal deoxynucleotidyltransferase dUTP nick-end labeling assay, enzyme-linked immunosorbent assay, and Western blot.
Results
Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by tadalafil treatment. Tadalafil also protected hearts against doxorubicin-induced cardiomyocyte atrophy/degeneration and myocardial fibrosis. No doxorubicin-induced apoptotic effects were seen between groups. Cardiac cGMP level was lower in the doxorubicin-treated group, however it was significantly increased with tadalafil treatment. Compared to the control group, the myocardial expression of 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin were significantly decreased in the doxorubicin-treated group, which were restored by the tadalafil treatment.
Conclusions
The present study indicates a protective effect of tadalafil mainly through cGMP signaling pathway against doxorubicin-induced nonischemic cardiomyopathy.
Keywords
Introduction
The antineoplastic drug doxorubicin (DOX) is widely used in the therapy of various malignant tumors including leukemia, lymphomas, and solid tumors such as ovarian, breast, lung, cervical, and uterine cancers [
[1]
]. However, its clinical use is limited by its adverse side effects: irreversible degenerative cardiomyopathy and congestive heart failure [2
, 3
]. The DOX-induced acute cardiotoxicity is characterized by hypotension, arrhythmia, and tachycardia while the chronic effects are manifested as cardiac dysfunction eventually leading to congestive heart failure [4
, 5
]. The development of cumulative dose-dependent cardiomyopathy may occur many years after the cessation of DOX treatment [6
, 7
]. Despite various therapeutic interventions adopted to protect the heart against DOX-induced cardiotoxicity, the deterioration in cardiac functions is often accompanied by high mortality rates. So far, the ability of these treatments to protect the heart from DOX-induced damage has been limited. Hence, there is an ongoing need to further investigate and develop efficient therapeutic agents to combat DOX-induced cardiac damage.Tadalafil (Tad) is a potent long-acting selective inhibitor of cGMP-specific phosphodiesterase-5 (PDE5), which hydrolyzes and eliminates cGMP in cells [
[8]
]. A previous study has shown that the long-acting PDE5 inhibitor, Tad, induces sustained cardioprotection against lethal ischemic injury [[9]
]. Several studies suggest that PDE5 inhibitors induce powerful beneficial effects for pulmonary arterial hypertension and ischemia/reperfusion injury [10
, 11
]. Our most recent study suggests that prolonged inhibition of PDE5 through short hairpin RNA interference would relieve cardiac remodeling and dysfunction following myocardial infarction [[12]
]. There has been only one study reporting benefits of Tad on nonischemic cardiomyopathy [[13]
], thus little is known on the mechanisms for Tad to exert such effects. It would be worth confirming the beneficial effects of Tad on DOX-induced cardiomyopathy. In the present study, we examined the effects of Tad on DOX-induced cardiomyopathy and investigated the specific mechanisms of its effects.Materials and methods
This study was approved by our Institutional Animal Research Committee and conformed to the animal care guidelines of the American Physiological Society. Cardiomyopathy was induced in male 10-week-old C57BL/6J mice (Dalian Medical University, Dalian, China) by a single intraperitoneal injection of DOX (Sigma, St. Louis, MO, USA) at a dose of 15 mg/kg, which has been reported to be cardiotoxic [
[14]
]. The presence of DOX-induced cardiomyopathy was confirmed both functionally and histologically in all mice that were given no therapeutic intervention by the observation of decreased left ventricular (LV) function (by echocardiography and cardiac catheterization). In sham-treated mice, the same volume of saline was injected in a similar manner. Tad (Lilly, Indianapolis, IN, USA) tablets were crushed in water and given to animals orally via gauge needle at a dose of 4 mg/kg/day for successive 14 days. This dose was chosen based on the interspecies dose extrapolation scaling to result in plasma concentrations equivalent to a human dose of 20 mg/day [[15]
]. Untreated control groups were given the same volume of saline.Mice were randomly assigned to receive (a) saline alone (Con group, n = 10); (b) DOX alone (DOX group, n = 16); (c) DOX plus Tad (DOX + Tad group, n = 16), or (d) Tad alone (Tad group, n = 10). Two weeks later, all surviving mice were killed with an overdose of pentobarbital after physiological examination. The hearts were excised and weighed, cardiac specimens were then subjected to histological, immunohistochemical, and molecular biological analyses.
Physiological studies
Animals were anesthetized via intraperitoneal injection with pentobarbital. Echocardiograms were then recorded with an echocardiographic system (Aloka) equipped with a 7.5-MHz imaging transducer as reported previously [
[16]
]. LV end-systolic diameter (LVDs) and end-diastolic diameter (LVDd) were measured from at least three consecutive cardiac cycles. After cardiac echocardiography, the right carotid artery was cannulated with a micromanometer-tipped catheter (SPR 407; Millar Instruments, Houston, TX, USA) and advanced into the aorta and then into the LV to record pressure and ±dP/dt.- Li Y.
- Takemura G.
- Kosai K.
- Yuge K.
- Nagano S.
- Esaki M.
- Goto K.
- Takahashi T.
- Hayakawa K.
- Koda M.
- Kawase Y.
- Maruyama R.
- Okada H.
- Minatoguchi S.
- Mizuguchi H.
- et al.
Postinfarction treatment with an adenoviral vector expressing hepatocyte growth factor relieves chronic left ventricular remodeling and dysfunction in mice.
Circulation. 2003; 107: 2499-2506
Histological analysis
After the echocardiography, each heart was removed and cut into 2 transverse slices. One was fixed in 10% buffered formalin and embedded in paraffin, after which 4-μm-thick sections were stained with hematoxylin–eosin (HE) or Masson. Quantitative assessments, including cell size and fibrotic area, were performed with a multipurpose color image processor (Image Pro Plus) with 20 randomly chosen high-power fields in each heart.
Enzyme-linked immunoassay
Levels of cGMP in the myocardium were assayed with an enzyme-linked immunosorbent assay (ELISA) (CycLex, Nagano, Japan). Three hearts from each group were used for this assay.
In situ nick end-labeling
Terminal dUTP nick end-labeling (TUNEL) assays were performed in sections with an ApopTag kit (Intergene, Purchase, NY, USA) mainly according to the instructions of the supplier. Mouse mammary tissue served as a positive control.
Western blotting
Lysates/proteins from heart tissues were used for Western blot analysis. Proteins were separated and transferred to membranes by standard protocols, after which they were probed with antibodies against myosin heavy chain (MHC), troponin I (both from Santa Cruz, Dallas, TX, USA), desmin (Sigma). Three to five hearts from each group were subjected to the blotting. The blots were visualized by means of chemiluminescence (ECL, Thermo, Rockford, IL, USA), and the signals were quantified by densitometry. a-Tubulin (analyzed with an antibody from Thermo) served as the loading control.
Statistical analysis
Statistical analysis was performed by using software of SPSS17.0 (Cary, NC, USA). Values are shown as mean ± SEM. The significance of differences between groups was evaluated with 1-way ANOVA followed by the Newman–Keuls multiple comparison test. Values of p < 0.05 were considered significant.
Results
Survival rates and heart-to-body weight ratios
Two weeks later, we evaluated survival rates and heart-to-body weight ratios. Three of DOX and two of DOX + Tad mice were dead, no mouse was dead in the Con or Tad groups. Survival rates of the group treated with DOX + Tad (87.5%) exhibited increasing tendency compared with the DOX group (81.25%) during the 14-day experimental protocol, although this change was insignificant. The heart-to-body weight ratio was significantly increased in the DOX group, which was reversed by Tad (Table 1).
Table 1Heart-to-body weight ratios among the groups.
| Groups | n | Heart weights (g) | Body weights (g) | Heart-to-body weight ratios (%) |
|---|---|---|---|---|
| Con | 10 | 0.098 ± 0.08 | 26.42 ± 0.91 | 0.37 ± 0.019 |
| DOX | 13 | 0.089 ± 0.13 | 21.26 ± 1.15 | 0.42 ± 0.037 |
| DOX + Tad | 14 | 0.092 ± 0.13 | 25.33 ± 1.02 | 0.36 ± 0.046 |
| Tad | 10 | 0.092 ± 0.11 | 26.69 ± 1.16 | 0.34 ± 0.038 |
* p < 0.05 versus Con group.
# p < 0.05 versus DOX group.Con, control; DOX, doxorubicin; Tad, tadalafil.
Physiological studies
The results of our physiological studies are summarized in Fig. 1. Echocardiography and cardiac catheterization performed 2 weeks after DOX administration showed that mice receiving DOX alone had significant cardiac functional deterioration characterized by enlargement of the LV cavity and signs of decreased cardiac function, i.e. increased LV diameter and end-diastolic pressure and decreased LV ejection fraction and ±dP/dt, compared with sham animals. Treatment with Tad significantly mitigated the DOX-induced impairment of cardiac function. Administration of Tad to sham animals had no effect on cardiac function.
Fig. 1Effects of tadalafil (Tad) on left ventricular (LV) geometry and function assessed 2 weeks after doxorubicin (DOX) injection using echocardiography and cardiac catheterization. Con, control; LVDd, LV diastolic diameter; LVDs, LV systolic diameter; %EF, percent ejection fraction; LVSP, LV systolic pressure; LVEDP, LV end-diastolic pressure; HR, heart rate. Bars are means ± SEM; *p < 0.05 versus Con group; #p < 0.05 versus DOX group.
Histological analysis
Examination of transverse sections of hearts stained with H&E (Fig. 2A) revealed that the transverse diameters of cardiomyocytes from the group receiving DOX alone were significantly smaller than in the sham group (13.78 ± 0.36 μm versus 14.42 ± 0.46 μm, p < 0.05), and that Tad exerted a significant protective effect against DOX-induced atrophy (transverse diameter 14.24 ± 0.39 μm; Fig. 2B). We then assessed cardiac fibrosis using Masson's trichrome staining sections (Fig. 2A), we found that the amount of fibrosis was significantly higher in the group receiving DOX alone than in the sham group (5.16 ± 0.36% versus 1.39 ± 0.22%, p < 0.05) and that the DOX-induced fibrosis was significantly reduced by Tad (2.35 ± 0.21%; Fig. 2B).
Fig. 2Effects of Tad on cardiac histology in mice 2 weeks after DOX injection. (A) Photomicrographs of histological hematoxylin–eosin (HE) and Masson's trichrome staining of heart specimens. (B) Graphs showing morphometric data. Bars are means ± SEM; *p < 0.05 versus Con group; #p < 0.05 versus DOX group. (C) Graphs show the terminal dUTP nick end-labeling assay-based apoptotic index among cardiomyocytes. Bars are means ± SEM; p = ns. Con, control; DOX, doxorubicin; Tad, tadalafil.
TUNEL-positive cells
TUNEL-positive cells were observed among cardiomyocytes from all 4 groups, but we found no significant difference in the incidence of TUNEL-positive cells between mice that received DOX and those that did not, and Tad had no significant effect on the incidence of TUNEL-positive cells (Fig. 2C). These results indicate that apoptosis is not involved in the present model of DOX-induced cardiomyopathy.
Cardiac cGMP level
Treatment with DOX decreased cGMP levels in the heart compared with the saline-treated control (Fig. 3A). The combined treatment with Tad and DOX augmented cGMP levels compared with DOX alone.
Fig. 3Enzyme-linked immunosorbent assay (ELISA) and Western analysis of the effects of Tad on myocardial expression of cGMP and three sarcomeric proteins: MHC, troponin I, and desmin. (A) Levels of cGMP in the myocardium were assayed with an ELISA. Bars are means ± SEM; *p < 0.05 versus Con group; #p < 0.05 versus DOX group. (B and C) Western blot analysis of effect of Tad on myocardial expression of 3 sarcomeric proteins, MHC, troponin I, and desmin. Bars are means ± SEM; *p < 0.05 versus Con group; #p < 0.05 versus DOX group. Con, control; DOX, doxorubicin; Tad, tadalafil; MHC, myosin heavy chain.
Expression of sarcomeric proteins
We found myocardial levels of 3 sarcomeric proteins, MHC, troponin I, and desmin, were significantly downregulated by DOX. The inhibitory effect of DOX on the expression of all 3 of these proteins was completely reversed by Tad (Fig. 3B and C).
Discussion
We used an oral administration regimen of Tad (4 mg/kg/day for 14 days), which is similar to reported levels in human subjects taking clinically relevant doses of Tad (20 mg p.o. daily for 1 week) [
[15]
].The present study provides the important evidence of the beneficial effects of Tad on cardiac dysfunction resulting from DOX-induced cardiomyopathy, a nonischemic cardiomyopathy. The prominent interesting findings were that Tad prevented DOX-induced atrophic degeneration of cardiomyocytes and cardiac fibrosis. Our findings suggest that several factors contribute to the cardioprotective effects of Tad against DOX-induced cardiomyopathy. The first is that Tad exerts an anti-atrophic/degenerative effect on cardiomyocytes. Sarcomeric proteins, including MHC, troponin I, and desmin, are important for the structural integrity and function of cardiomyocytes, and their myocardial expression is reportedly downregulated by DOX [
[17]
], an effect we confirmed in the present study. Our new finding is that Tad significantly restores the expression of sarcomeric proteins in the presence of DOX.In the present study, we also observed that DOX stimulates myocardial fibrosis, which is consistent with the previous study [
[18]
] and that Tad prevents the pathological process. Because myocardial fibrosis contributes to both systolic and diastolic dysfunction [- Esaki M.
- Takemura G.
- Kosai K.
- Takahashi T.
- Miyata S.
- Li L.
- Goto K.
- Maruyama R.
- Okada H.
- Kanamori H.
- Ogino A.
- Ushikoshi H.
- Minatoguchi S.
- Fujiwara T.
- Fujiwara H.
Treatment with an adenoviral vector encoding hepatocyte growth factor mitigates established cardiac dysfunction in doxorubicin-induced cardiomyopathy.
Am J Physiol Heart Circ Physiol. 2008; 294: H1048-H1057
5
, 19
], its reduction is likely another important way in which Tad may mitigate LV remodeling and heart failure.The exact pathogenesis of DOX-induced cardiotoxicity is still not entirely clear although a diverse set of mechanisms have been proposed, including oxidative stress, mitochondrial DNA damage, intracellular calcium overload, cytokine release, cardiomyocyte apoptosis and cardiomyocyte atrophy [
20
, 21
, 22
, 23
, 3
, 24
]. A previous finding indicates that apoptosis among cardiomyocytes is a leading cause of cardiac dysfunction in DOX-induced cardiomyopathy [[25]
]. Seeking evidence of DOX-induced apoptosis, we conducted a series of TUNEL assays but detected no effect of DOX or Tad on the incidence of apoptosis. Thus, our findings suggest that cardiomyocyte apoptosis is not important for disease progression in the present model. It has been reported that cardiomyocyte atrophy plays a major role in the DOX-induced cardiomyopathy [[24]
]. In our experiment, we confirmed that DOX significantly induced cardiomyocyte atrophy. Our important finding was that Tad significantly attenuated DOX-induced cardiomyocyte atrophy. We speculate that cardiomyocyte atrophy is a main reason to cause DOX-induced impairment of cardiac function, and that Tad plays a protective role by mitigating the DOX-induced cardiomyocyte atrophy.cGMP is an intracellular second messenger that mediates multiple tissue and cellular responses, including its role in late-phase preconditioning [
26
, 27
]. cGMP plays important roles in the regulation of smooth muscle relaxation, platelet aggregation, intestinal secretion, and endochondrial ossification through the activation of cGMP-dependent protein kinase [[28]
]. Under physiological conditions, cGMP is inactivated via hydrolytic degradation by PDE. PDE5 negatively regulates intracellular cGMP level and its activity [[29]
]. PDE5 inhibitors have been observed to play a critical role against cardiac ischemia-reperfusion injuries by activating cGMP/cyclic GMP-dependent protein kinase (PKG) signaling pathway [[30]
]. The PDE5A inhibitor, sildenafil, can prevent myocyte hypertrophy, and improve heart function of mice exposed to chronic pressure overload induced by transverse aortic constriction through blocking the intrinsic catabolism of cGMP [[31]
]. However, the role of cGMP/PKG signaling in protection against DOX-induced cardiotoxicity is not well-known yet.A previous study has found that cGMP can promote rat glial cells and microglia MHC expression [
[32]
]. It has been reported that PDE5 inhibition prompted troponin I phosphorylation through PKG-dependent cGMP signaling pathway [[33]
]. In the present study, we found that Tad significantly restores, with accompaniment of cGMP increase, the myocardial expression of MHC, troponin I, and desmin which were decreased in the DOX group. We speculate that Tad upregulates the expression of myocardial MHC, troponin I, and desmin through activating cGMP signaling pathway, that finally prevents cardiomyocyte atrophy and degeneration induced by DOX. A previous study has found that myocardial fibrosis associated with the increased expression of transforming growth factor (TGF)-β1 and cGMP can reduce cardiac fibrosis through inhibiting expression of TGF-β1 [[34]
]. In our study, we observed a significant increase in cGMP levels and decrease in fibrosis in the hearts of mice treated with DOX + Tad compared to DOX alone. Fibrosis is considered to be a common pathological change of cardiac remodeling and heart failure; inhibition of myocardial fibrosis leads to the improvement of cardiac function. The present study indicates that Tad reduces DOX-induced myocardial fibrosis possibly through up-regulation of cGMP concentration. We therefore suggest that Tad exerts its beneficial effects via the cGMP signaling pathway (Fig. 4), which was otherwise inhibited in the DOX group.
Fig. 4Proposed scheme showing possible mechanisms for the protective effects of tadalafil against doxorubicin-induced nonischemic cardiomyopathy. PDE5, phosphodiesterase 5; PKG, cyclic GMP-dependent protein kinase; TGF, transforming growth factor; MHC, myosin heavy chain; TnI, troponin I.
Conclusion
The present study suggests the protective effects of Tad against DOX-induced cardiomyopathy, mitigating DOX-induced impairment of cardiac function in mice, significantly attenuating DOX-induced atrophic degeneration of cardiomyocyte and myocardial fibrosis possibly through the cGMP signaling pathway.
Conflict of interest
There is no conflict of interest in this work.
Acknowledgments
We thank Meilan Wang and Ailin Song for technical assistance. This work was supported by National Natural Science Foundation of China , No. 81170187 (to L. Li).
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Article Info
Publication History
Published online: June 03, 2013
Accepted:
March 21,
2013
Received in revised form:
March 14,
2013
Received:
January 28,
2013
Identification
Copyright
© 2013 Japanese College of Cardiology. Published by Elsevier Inc.
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