Oxidative Stress Regulates Left Ventricular PDE5 Expression in the Failing Heart
Background— Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF.
Methods and Results— Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)-induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased ≈4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3′-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF.
Conclusions— Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.
Received September 3, 2009; accepted February 5, 2010.
Congestive heart failure (CHF) is the leading cause of mortality in developed countries and continues to increase in prevalence. Phosphodiesterase type 5 (PDE5) selectively hydrolyzes cyclic 3′,5′-guanosine monophosphate (cGMP), and selective inhibition of PDE5 can increase cGMP bioavailability. It is generally believed that PDE5 is not present in normal cardiac myocytes, so that selective PDE5 inhibition has no direct inotropic effect in normal hearts.1 However, recent work by Kass et al demonstrated that selective inhibition of PDE5 with sildenafil markedly attenuated the left ventricular (LV) hypertrophy and dysfunction produced by chronic pressure overload secondary to transverse aortic constriction (TAC) in mice.2 Thus PDE5 inhibition has also been reported to attenuate myocardial infarct-induced LV remodeling3 and LV hypertrophy produced by chronic isoproterenol infusion in rats.4 In addition, PDE5 expression was increased in hypertrophied human right ventricles and left ventricles from humans with heart failure,5,6 and overexpression of PDE5 in cardiac myocytes exacerbated myocardial infarction-induced LV remodeling in mice.6 However, the regulation of PDE5 expression in cardiac myocytes of the failing heart is not clear.
Clinical Perspective on p 1483
In this study, we provide evidence that PDE5 protein content is increased in cardiac myocytes in heart failure samples from human or mouse and that expression of PDE5 in cardiac myocytes in the failing heart is regulated by myocardial oxidative stress. We found that the increase of myocardial PDE5 expression in human and mouse failing hearts was associated with myocardial 3′-nitrotyrosine and 4-hydroxynonenal (4-HNE) levels (markers of oxidative stress). Importantly, attenuation of oxidative stress with the superoxide dismutase (SOD) mimetic M40401 decreased TAC-induced PDE5 expression, ventricular hypertrophy, LV dysfunction, and pulmonary congestion. Conversely, increasing myocardial oxidative stress by deletion of the SOD3 gene in mice increased myocardial PDE5 expression and exacerbated TAC-induced LV hypertrophy and heart failure. In addition, selective inhibition of PDE5 with sildenafil attenuated the TAC-induced LV oxidative stress, hypertrophy, and dysfunction. Together, our study provides the first direct evidence that oxidative stress regulates PDE5 expression in cardiac myocytes.
Materials and Methods
The experimental studies in mice and human tissues were approved by the Institutional Animal Care and Use Committee and the Institutional Review Board at the University of Minnesota, respectively.
TAC Procedure and Experimental Treatments
Male C57BL/6 mice (Taconic, Germantown, NY) ≈2 months old were used for the TAC procedure. TAC was performed using a 27G needle to calibrate the degree of aortic constriction, as previously described.7,8 For the M40401 study, immediately after recovery from anesthesia after TAC, the mice were randomly assigned to 2 groups treated either with M40401 (10 mg/kg per day IP) or vehicle for 2 weeks. For the study in SOD3 knockout (KO) mice, PDE5 expression and TAC-induced ventricular remodeling were determined in SOD3 KO mice and control wild-type mice. For the sildenafil study, after TAC, mice were randomly assigned to 2 groups, 1 group treated with sildenafil (50 mg/kg twice a day for 2 weeks) by gavage and the other treated with vehicle.
Mouse echocardiographic images were obtained with a Visualsonics high-resolution Veve 660 or 770 system as previously described.8,9
Western Blots and Chemical and Histological Analysis
The detailed methods for sample collection, Western blot, immunostaining, PDE5 and protein kinase G (PKG) activity measurement, cell culture, and treatment are included in the online-only Data Supplement.
Data in this study were first analyzed to determine whether data were normally distributed using normality test (Kolmogorov-Smirnov) provided by Sigmastat. If data were normally distributed, the data were presented as mean±SE. If the data were not normally distributed, the data were presented as median (±SE).
Data of 2 groups were compared with unpaired t test. For the effect of M40401 or sildenafil on TAC-induced LV remodeling, 1-way ANOVA was used to test for differences among treatment groups, followed by pairwise multiple comparisons with the Fisher least significant difference method. If the data were not normally distributed, rank-based 1-way ANOVA was performed to test for differences among treatment groups, followed by pairwise multiple comparisons with the Dunn method. Two-way ANOVA was used to test for differences between inducible nitric oxide synthase (iNOS) KO and wild-type mice under control conditions and after TAC. In addition, 2-way ANOVA was also used to test for differences between SOD3 KO and wild-type mice under control conditions and after TAC. For 2-way ANOVA, if ANOVA demonstrated a significant effect, all pairwise multiple comparisons were made with the Turkey method under the interaction term. Statistical significance was defined as P<0.05.
Increased PDE5 Expression in Human Heart Failure Samples Is Correlated With Myocardial Oxidative Stress
We studied LV PDE5 expression in 6 patients with CHF and 6 normal donor hearts (online-only Data Supplement Table I). PDE5 expression was increased in all CHF hearts, with the mean signal intensity increased more than 4.5-fold as compared with the donor hearts (Figure 1A and 1B). Myocardial atrial natriuretic peptide (ANP) protein, a marker associated with ventricular hypertrophy and heart failure, was also significantly increased in CHF hearts (Figure 1A and 1B). Because previous studies indicate that oxidative stress can induce PDE5 expression in smooth muscle cells, levels of 3′-nitrotyrosine and 4-HNE were determined in the myocardial samples. Both 3′-nitrotyrosine and 4-HNE were significantly increased in the human CHF samples (Figure 1A and 1B). The increase of myocardial PDE5 was significantly correlated to the increase of 3′-nitrotyrosine and 4-HNE (Figure 1C and 1D).
LV samples from 5 donor hearts and 5 failing hearts used for the Western blots were also subjected to histological staining for PDE5 expression. Consistent with previous reports in normal human heart tissue, we found PDE5 predominantly expressed in vascular smooth muscle cells, with minimal expression in cardiac myocytes (online-only Data Supplement Figure I). Interestingly, in the samples from failing hearts, PDE5 was expressed in both cardiac myocytes and vascular smooth muscle cells.
Real-time quantitative polymerase chain reaction demonstrated that PDE5 messenger RNA (mRNA) was significantly increased 2.4-fold in the human heart failure samples (Figure 1E), and ANP mRNA was increased 42-fold in the human heart failure samples (Figure 1F).
SOD Mimetic M40401 Attenuated Myocardial Oxidative Stress and PDE5 Expression in Pressure-Overloaded Hearts
Because PDE5 expression in the human heart samples was significantly correlated with 3′-nitrotyrosine and 4-HNE content, we speculated that pressure overload might increase both ventricular oxidative stress and PDE5 expression and that decreasing the oxidative stress might attenuate PDE5 expression and LV dysfunction. To test this hypothesis, wild-type mice were subjected to severe pressure overload using the TAC procedure and immediately randomized to treatment either with the SOD mimetic M40401 or vehicle. Ventricular structure and function were determined 2 weeks after TAC.
The increase in LV systolic pressure produced by TAC resulted in LV hypertrophy and systolic dysfunction. The LV hypertrophy and systolic dysfunction causes an increase in LV diastolic pressure that later results in left atrial (LA) hypertrophy and ultimately pulmonary congestion. Therefore, TAC-induced cardiac remodeling can be evaluated by the progressive development of LV and LA hypertrophy and increased lung weight.
Two weeks after TAC, the ratio of LV weight to body weight was increased by 81%, the ratio of LA weight to body weight increased by 197%, and the ratio of lung weight to body weight increased by 77% (Figure 2A through 2D). TAC also caused significant increases in tissue weight of LV, LA, and lung and their ratio to tibial length (online-only Data Supplement Table II). TAC also caused a significant decrease of LV ejection fraction and an increase of LV end-systolic diameter (Figure 2E and 2F; online-only Data Supplement Table III). These changes were associated with significant increases of ventricular ANP, PDE5, and the oxidative stress markers 3′-nitrotyrosine and 4HNE (Figure 3A and 3B). Histological staining demonstrated that TAC caused increased PDE5 protein expression in cardiac myocytes and vascular smooth muscle (Figure 3C). Scavenging superoxide with the SOD mimetic, M40401, significantly attenuated the TAC-induced increases of LV, LA, and lung weight (online-only Data Supplement Table II) and their ratio to body weight or tibial length (Figure 2A through 2D; online-only Data Supplement Table II). M40401 also significantly attenuated the TAC-induced decrease of LV ejection fraction (Figure 2E and 2F) and the increase of the LV end-systolic diameter (online-only Data Supplement Table III). M40401 significantly attenuated the TAC-induced increase of ventricular ANP, PDE5, and the oxidative stress markers 3′-nitrotyrosine and 4-HNE (Figure 3A and 3B).
Real-time polymerase chain reaction showed that myocardial PDE5 mRNA was moderately increased as compared with the sham control group (P<0.05) in the animals exposed to TAC and that TAC-induced increase of PDE5 was not statistically different after M40401 treatment (online-only Data Supplement Figure II). In addition, the overall increase of PDE5 mRNA in isolated cardiac myocytes from heart failure mice is similar to its increase in the myocardial tissues (online-only Data Supplement Figure II).
M40401 Had No Significant Effect on PKG Activity and Phosphorylation of Vasodilator-Stimulated Protein
cGMP causes PKG phosphorylation, and phosphorylated PKG induces phosphorylation of vasodilator-stimulated protein at site of Ser239. Therefore, p-VASPSer239 is often used as an indicator of PKG activity. We found that severe TAC caused a significant increase of p-VASPSer239 expression (Figure 3G and 3H), which is in agreement with previous report from Kass et al10 in the same model. M40401 also had no significant effect on TAC-induced increase of p-VASPSer239 expression (Figure 3G and 3H), which is consistent with myocardial PKG activity in these mice. These data suggest that PDE5 expression is not affected by PKG activity.
M40401 Attenuated TAC-Induced Phosphorylation of Extracellular Signal-Regulated Kinase and Akt
TAC-induced LV oxidative stress, hypertrophy, and heart failure are often associated with increased activation of extracellular signal-regulated kinase (ERK), Akt, and glycogen synthase kinase 3β (GSK-3β). We found that chronic TAC caused significant increase of p-ERKThr202/204, p-AktSer473, and p-GSK-3βSer21/9, but not their total protein contents (Figure 4). M40401 significantly attenuated TAC-induced increase of p-ERKThr202/204, p-AktSer473, and p-GSK-3βSer21/9. These results are consistent with the decreased LV oxidative stress after M40401.
iNOS Gene Deletion Attenuated TAC-Induced Myocardial Oxidative Stress and PDE5 Expression
Recently, we demonstrated that iNOS gene deletion (iNOS−/–) significantly attenuated TAC-induced myocardial oxidative stress, LV hypertrophy, and heart failure.11 We therefore speculated that the decrease of myocardial oxidative stress in iNOS−/– mice after TAC would consequently attenuate myocardial PDE5 expression in these mice. Consequently, LV ANP, PDE5, 3′-nitrotyrosine, and 4-HNE expressions were determined in iNOS−/– hearts and wild-type hearts under control conditions and after TAC for 2 weeks. As previously reported, LV ANP, 3′-nitrotyrosine, and 4-HNE expressions were all significantly increased in both wild-type and iNOS−/– hearts,11 whereas iNOS−/– significantly attenuated TAC-induced increases of LV ANP, 3′-nitrotyrosine, and 4-HNE expression (Figure 5). As anticipated, TAC caused significant increases of LV PDE5 expression in wild-type and iNOS−/– hearts, whereas iNOS−/– significantly attenuated TAC-induced increase of LV PDE5 expression (Figure 5). The relatively small decreases of LV 3′-NT and 4-HNE in iNOS−/– mice after TAC suggest that iNOS or iNOS-derived NO might play an important role for the PDE5 expression in the failing hearts.
SOD3 Gene Deletion Increased Myocardial PDE5 Expression and Exacerbated TAC-Induced LV Dysfunction
We previously demonstrated that SOD3 gene deletion (SOD3−/–) mice had mildly increased myocardial oxidative stress under basal conditions, but markedly enhanced TAC-induced LV oxidative stress, hypertrophy, and dysfunction.12 We subsequently hypothesized that the SOD3−/– heart would have exacerbated TAC-induced PDE5 expression. To test this hypothesis, wild-type control mice and mice with SOD3−/– were subjected to TAC procedure for 2 weeks, and ventricular structure and function were determined. Consistent with our previous report,12 SOD3−/– has negligible effects on LV weight, lung weight, and their ratio to body weight or tibial length (Figure 6A and 6B; online-only Data Supplement Table IV) under control conditions. SOD3−/– had no effect on LV function under control conditions (Figure 6C; online-only Data Supplement Table V). However, the SOD3−/– had significantly exacerbated TAC-induced cardiac remodeling, as demonstrated by significant increases of LV weight and lung weight ratios to body weight or tibial length (Figure 6A and 6B; online-only Data Supplement Table IV). After TAC, the SOD3−/– heart had significantly decreased LV ejection fraction, a dramatic increase of LV end-systolic diameter, and an increase of LV end-diastolic diameter (Figure 6C; online-only Data Supplement Table V).
Interestingly, under control conditions, although the SOD3−/– heart had only moderately increased 4-HNE expression and did not significantly increase myocardial 3′-NT expression, myocardial PDE5 expression was significantly increased by 69% in these mice. As anticipated, the SOD3−/– heart had a significantly greater increase of ventricular ANP, 3′-nitrotyrosine, 4HNE, and PDE5 expression after TAC (Figure 6D and 6E). The greater increase of LV PDE5 in SOD3−/– mice after TAC appears to be an additive effect.
Selective PDE5 Inhibition Attenuated TAC-Induced LV Dysfunction in Wild-Type Mice
Because the TAC-induced LV dysfunction was associated with increased myocardial PDE5 RNA and protein expression, we determined the effect of PDE5 inhibition with sildenafil on TAC-induced ventricular hypertrophy and dysfunction. Briefly, mice after TAC were divided into 2 groups treated either with sildenafil or vehicle. Two weeks after TAC, mice in the vehicle-treated group had significant increases in the LV, LA, and lung weight (online-only Data Supplement Table VI), ratio to body weight (Figure 7A through 7D), or tibial length (online-only Data Supplement Table VI). TAC significantly decreased LV ejection fraction, increased LV end-systolic diameter (Figure 7E; online-only Data Supplement Table VII), and decreased LV maximum rate of rise of pressure (dP/dtmax) and LV maximum rate of decline of pressure (dP/dtmin) in the vehicle-treated group (online-only Data Supplement Table VII). Ventricular ANP, PDE5, and the oxidative markers 3′-nitrotyrosine and 4-HNE were also significantly increased after TAC (Figure 8A and 8B). Consistent with previous reports from Kass et al,2 sildenafil significantly attenuated the TAC-induced LV hypertrophy and cardiac function as demonstrated in Figure 7 and online-only Data Supplement Tables VI and VII. Interestingly, PDE5 inhibition with sildenafil also significantly attenuated the TAC-induced increases of ANP, 3′-nitrotyrosine, 4-HNE, and PDE5 protein expression (Figure 8A and 8B).
Selective PDE5 Inhibition Attenuated TAC-Induced LV Hypertrophy and Dysfunction in SOD3−/– Mice
Because LV PDE5 expression was increased in SOD3−/– mice, we studied the effect of PDE5 inhibition on TAC-induced LV hypertrophy and dysfunction. Results showed that sildenafil significantly attenuated TAC-induced LV hypertrophy and pulmonary congestion, decreased LV ejection fraction, and decreased LV contractility in SOD3−/– mice (online-only Data Supplement Table VIII, Table IX, and Figure III).
Global Gene Profiling Indicates That Myocardial PDE5 mRNA Was Moderately Increased in Heart Failure Human LV Samples
To understand the alteration of myocardial PDE5 mRNA in human heart failure samples, we downloaded an original microarray data set (GDS651; Reference Series: GSE1145) used for investigation of global gene profiles of LV samples from normal donor hearts (n=11) and hearts with idiopathic dilated cardiomyopathy (n=15) or ischemic cardiomyopathy (n=11). These microarray data include 3 probe sets for PDE5 mRNA. The 3 probe sets revealed that LV PDE5 mRNA was significantly increased 1.3-, 1.7-, or 1.9-fold in hearts with idiopathic dilated cardiomyopathy (online-only Data Supplement Figure IV). Two probe sets revealed that LV PDE5 mRNA was significantly increased 1.6- and 2.1-fold in hearts with ischemic cardiomyopathy (online-only Data Supplement Figure IV). These data suggest that increased PDE5 mRNA may be a common phenomenon in both ischemic and idiopathic dilated cardiomyopathy.
Effect of SOD Mimetic on Attenuation of PDE5 Expression in Cultured Cardiomyocytes Was Unaffected by PKG Inhibition
To further confirm that PKG activity had no effect on PDE5 expression, we subsequently determined the effect of SOD mimetic MnTMPyP on angiotensin-II induced PDE5 expression in cultured cardiac myocytes with or without PKG inhibitor. Angiotensin-II increased 3′-NT, PDE5, and ANP expression in cardiac myocytes. SOD mimetic MnTMPyP significantly attenuated angiotensin-II induced increase of ANP expression in cultured cardiac myocytes, whereas the above effect was abolished by addition of PKG inhibitor KT5832 (online-only Data Supplement Figure V). SOD mimetic MnTMPyP also significantly attenuated angiotensin-II-induced expression of 3′-NT and PDE5 in cultured cardiac myocytes. However, addition of PKG inhibitor KT5832 after MnTMPyP had no effect on expression of 3′-NT and PDE5 in cardiac myocytes (online-only Data Supplement Figure V), indicating that the attenuation of PDE5 expression by reduction of oxidative stress was not PKG-dependent.
To the best of our knowledge, this represents the first demonstration that oxidative stress regulates PDE5 protein expression in cardiac myocytes in the failing hearts. We demonstrated that increased myocardial PDE5 expression in the failing hearts is significantly correlated with increases of the ventricular oxidative stress markers 3′-nitrotyrosine and 4-HNE. Subsequently, we demonstrated that LV oxidative stress and PDE5 expression were both increased in TAC-induced heart failure in mice, and decreasing the TAC-induced ventricular oxidative stress with the SOD mimetic M40401 resulted in lower PDE5 expression and improved LV dysfunction. Moreover, we demonstrated that iNOS gene deletion attenuated TAC-induced myocardial oxidative stress and PDE5 expression. Conversely, we found that increased ventricular oxidative stress in SOD3 KO mice was associated with increased ventricular PDE5 expression and significantly exacerbated TAC-induced LV hypertrophy and dysfunction. Finally, PDE5 inhibition with sildenafil diminished the TAC-induced ventricular oxidative stress, PDE5 expression, LV hypertrophy, and LV dysfunction. Together, these data demonstrated that oxidative stress regulates the PDE5 expression in cardiac myocytes in the failing hearts, and the increased PDE5 expression in the failing heart contributes to the adverse ventricular remodeling in the failing hearts. The newly identified relationship between myocardial oxidative stress and PDE5 in the failing hearts suggests that both antioxidant(s) and PDE5 inhibitor(s) may be promising therapeutic approaches for attenuating myocardial oxidative stress, PDE5 expression, and PDE5 activity.
One of the interesting findings in the present study is the increased PDE5 expression in cardiac myocytes in both human and mouse failing heart. After the initial submission of this manuscript, another study also reported that PDE5 expression was increased in cardiac myocytes in failing human hearts.6 This finding is important, as previous studies have demonstrated that PDE5 expression is very low in cardiac myocytes under basal conditions,1 and on the basis of this finding, the prevailing hypothesis was that PDE5 inhibition would have minimal effects on cardiac function in diseased hearts.1 However, studies pioneered by Kass et al demonstrated that selective PDE5 inhibition with sildenafil profoundly attenuates chronic pressure overload-induced LV hypertrophy and dysfunction in mice subjected to TAC.2 Our results in the present study further demonstrate increased PDE5 expression in cardiac myocytes in the failing human and mouse heart. These results support and extend the previous work from Kass et al and support the use of PDE5 inhibitors as a novel approach to treat heart failure. Additionally, recent studies showed that PDE5 activity was increased in LV tissues obtained from heart failure mice,2 as well as in multiple tissues obtained from pacing-induced CHF in the dog model.13 This suggests that the increase of PDE5 protein and activity in heart failure subjects appears to be a common phenomenon across multiple species and may even extend beyond the myocardial tissues.
The finding that M40401 reduced LV oxidative stress, hypertrophy, and ventricular dysfunction in mice exposed to TAC is of considerable interest. M40401 is a potent, highly diffusible SOD mimetic14,15 that has been shown to be effective in attenuating oxidative stress in many experimental models, including shock, ischemia-reperfusion, and inflammation.16 M40403, a close analogue of M40401, is the only SOD mimetic advanced into phase II clinical trials. We found that M40401 moderately improved coronary blood flow in the CHF dog model and enhanced acetylcholine-induced coronary vasodilatation in dogs with pacing-induced heart failure.17 Although no previous study has determined the effect of M40401 on the chronic systolic overload-induced heart failure, the finding that M40401 attenuated LV hypertrophy and dysfunction in the mice exposed to TAC is conceptually consistent with previous reports that decreasing oxidative stress attenuates LV hypertrophy and dysfunction produced by chronic systolic overload.18–20 Thus SOD mimetic Mn-TBAP has been reported to attenuate both myocardial oxidative stress and LV dysfunction in peroxisome proliferator-activated receptor γ deficient mice,21 whereas administration of the antioxidant pyrrolidine dithiocarbamate significantly attenuated TAC-induced LV oxidative stress and dysfunction in mice.22 The profound cardiac-protective effect of M40401 in attenuating TAC-induced heart failure warrants additional studies in different or large animal models.
The findings that sildenafil attenuated the TAC-induced increases of LV mass, LA mass, lung weight, and right ventricular weight indicate that PDE5 inhibition attenuated TAC-induced ventricular dysfunction and cardiac remodeling. The findings of higher LV ejection fraction, LV systolic pressure, LV dP/dtmax, and LV dP/dtmin in the sildenafil-treated group indicate that PDE5 inhibition improved function of the chronically overloaded LV and are consistent with the previous report from Kass et al.2 In addition to attenuating the TAC-induced LV hypertrophy and dysfunction in mice, sildenafil also lessened the TAC-induced increases of myocardial PDE5, 3′-nitrotyrosine, and 4-HNE, suggesting that increased PDE5 activity in the failing heart might contribute to the increased oxidative stress and the upregulation of PDE5. The decreased myocardial oxidative stress after sildenafil may be a result of decreased LV remodeling after PDE5 inhibition2 or may result from its capacity in scavenging superoxide anion or inhibition of H2O2 generation, as recently reported by Fernandes et al.23
It is well established that oxidative stress is increased and contributes to cardiac hypertrophy.24 The finding that oxidative stress regulates LV PDE5 expression and that this relationship may drive hypertrophy and heart failure is novel. The increased myocardial oxidative stress in response to pressure overload may arise from several sources, including mitochondrial electron transport leakage,18 increases of nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase25,26 and xanthine oxidase,27 uncoupled NO synthase,11,19 or decreased antioxidant expression (such as SOD312,28,29 or SOD1). The content of 3′-nitrotyrosine and 4-HNE are often used as tissue oxidative stress markers, and the increase of myocardial oxidative stress as demonstrated by increased 3′-nitrotyrosine and 4-HNE in the LV of mice after TAC is consistent with previous reports from this laboratory8,11,30 and others using the same experimental model.19 We recently demonstrated that iNOS KO significantly attenuated TAC-induced myocardial oxidative stress and heart failure.11 The greatly attenuated PDE5 expression and small reduction of 3′-NT in iNOS KO mice after TAC suggests that iNOS or iNOS-derived NO might play an important role for the increased PDE5 expression in the failing hearts. The finding that higher PDE5 expression in SOD3 KO under control conditions and after TAC is consistent with our previous reports that SOD3 plays an important role in regulating myocardial oxidative stress and protecting hearts against TAC or infarction-induced ventricular remodeling.12,31 The finding that iNOS gene deletion, SOD3 KO, and M40401 alter PDE5 expression in the failing hearts suggests that increase of PDE5 expression in the failing hearts is not limited to a particular source of increased oxidative stress.
M40401 attenuated myocardial PDE5 expression but had no effect on myocardial PKG activity and p-VASPSer239 expression. The unchanged myocardial PKG activity and p-VASP after M40401 in the failing hearts may reflect an overall balance of myocardial cGMP availability in cardiac myocytes. Specifically, decreased myocardial PDE5 expression after M40401 is anticipated to attenuate PDE5-dependent cGMP degradation, whereas decreased myocardial ANP (or B-type natriuretic peptide) after M40401 could simultaneously attenuate ANP or B-type natriuretic peptide-dependent cGMP production. Indeed, Kass et al recently reported that addition of BH4 (a NOS cofactor and antioxidant) significantly attenuated TAC-induced myocardial oxidative stress and heart failure in mice, but also had no effect on myocardial PKG activity or p-VASPser239 content.10 In the cultured myocytes, SOD mimetic attenuated angiotensin-induced oxidative stress and PDE5 expression in cultured myocytes, whereas the above effect was unaffected by PKG inhibitor KT5823. These data suggest that decreased PDE5 expression after antioxidant treatment may be through a PKG-independent pathway.
The significant increase of PDE5 expression in the human and mouse heart failure samples was associated with moderately increased PDE5 mRNA, suggesting that increased myocardial PDE5 protein in the failing heart is partially regulated at the transcriptional level. This mechanism is also supported by the finding of moderate increases of LV PDE5 mRNA in patients with idiopathic dilated cardiomyopathy or ischemic cardiomyopathy, as revealed by the global microarray data.
The present data demonstrate that PDE5 protein expression is increased in the failing heart and that this increase is related to the increased myocardial oxidative stress. However, because oxidative stress can broadly affect many signaling pathways and alter the expression and modification of many important molecules, the precise molecular mechanism by which oxidative stress can regulate PDE5 expression is yet to be fully defined.
In summary, our study demonstrated that myocardial oxidative stress causes increased PDE5 expression in the failing heart, and selective PDE5 inhibition or inhibition of oxidative stress induction of myocardial PDE5 expression using M40401 protected heart against pressure overload-induced LV hypertrophy and CHF.
The authors gratefully acknowledge the expert technical assistance provided by Jennifer L. Fricton and the Histology and Microscopy Core of the Lillehei Heart Institute.
Sources of Funding
This study was supported by National Heart, Lung, and Blood Institute Grants HL71790 (Dr Chen) from the National Institutes of Health. Drs Fassett, Hu, and Zhang are recipients of Scientist Development Award from American Heart Association National Center.
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Congestive heart failure (CHF) is a leading cause of mortality in developed countries that is increasing in prevalence. In the present study, we found that myocardial phosphodiesterase type 5 (PDE5) protein content is increased in patients with end-stage CHF and that this increase is correlated with markers of increased myocardial oxidative stress. Inhibition of PDE5 increases cyclic 3-guanosine monophosphate bioavailability and has been shown to attenuate systolic overload-induced left ventricular (LV) hypertrophy and CHF in animal models. Our finding that either decreasing oxidative stress with a superoxide dismutase mimetic or inducible nitric oxide synthase gene deletion blunted the increase of myocardial PDE5 expression and LV dysfunction in mice subjected to systolic pressure overload implies that increased oxidative stress and inducible nitric oxide synthase expression at least partially contributed to the increased PDE5 expression and contractile dysfunction. The finding that inhibition of PDE5 with sildenafil reduced myocardial oxidative stress, PDE5 expression, and LV dysfunction indicates a role for the increased PDE5 expression in contributing to the increased oxidative stress and LV dysfunction in the overloaded heart. These data provide the first direct evidence that oxidative stress and inducible nitric oxide synthase can regulate PDE5 expression in cardiac myocytes and suggest that PDE5 inhibition, antioxidant treatment, or inducible nitric oxide synthase inhibition can decrease PDE5 expression in the overloaded heart and might be effective approaches to attenuate systolic overload-induced LV hypertrophy and CHF. This work suggests that further exploration of the use of PDE5 inhibitors or antioxidants in CHF is warranted.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.906818/DC1.
↵*Drs Lu and Xu contributed equally to this work.