Introduction

Phosphodiesterase (PDE) type 5 selectively hydrolyzes cyclic guanosine monophosphates.1 An inhibitor of PDE5, sildenafil, leads to smooth muscle relaxation in the corpus cavernosum, which governs penile erection, and it is widely used to treat erectile dysfunction.2 Recent growing evidence supports the cardio-protective effects of sildenafil against ischemia reperfusion3, 4 and anthracycline toxicity,5 and PDE5 inhibition by sildenafil blunts responses to adrenergic-stimulated cardiac contractility.6 Sildenafil also suppresses chronic cardiac hypertrophy and dysfunction attributable to pressure or volume overload.7, 8

Udenafil is a novel, potent PDE5 inhibitor that exhibits a similar molecular structure to that of sildenafil. An in vitro study evaluating various PDE isoenzymes demonstrated that the selectivity of udenafil is comparable to sildenafil.9 Udenafil exhibits potent efficacy in erectile dysfunction with a broad safety margin. Therefore, it has been widely used in the treatment of erectile dysfunction since 2005.10 The similar molecular structures, mechanisms of action and potency of udenafil and sildenafil suggest that udenafil has cardio-protective effects. However, there is a lack of studies on the role of udenafil in the heart.

This study was performed to determine whether udenafil exhibits a protective effect on pressure-overload cardiac hypertrophy. We created a chronic pressure-overload cardiac hypertrophy model in rats using suprarenal abdominal aorta constriction (SAC) and investigated the effects of udenafil on the following factors: (1) prevention of left ventricular (LV) dysfunction and remodeling, (2) exercise capacity and (3) survival. We also sought to elucidate the mechanisms mediating the effects of udenafil.

Materials and Methods

Animals

A total of 100 male 8-week-old Sprague–Dawley rats, weighing ~300 g, were used for this study. Rats were divided into three groups: sham (n=24), suprarenal aortic constriction (SAC) (n=40) and SAC+udenafil (n=36). One, 8 and 7 rats in the sham, SAC and SAC+udenafil groups, respectively, died within 3 weeks post surgery. The mortality rates in the acute stage post surgery were 4.1% in the sham group and 19.7% in the SAC group. Rats with incomplete modeling, which was defined as LV septal wall thickness (LVSWT) by using transthoracic echocardiography <2.5 mm at the 3rd week of SAC in the SAC (n=9) and SAC+udenafil (n=11) groups were excluded. The modeling success rate was 67.2%. Four rats in each group were euthanized at the 3rd week post operation for baseline analyses, including myocardial histopathological assessment, hemodynamic study and serum cytokine measurement. Therefore, the final numbers of animals that had full follow-up between the 3rd and 20th weeks post operation were 19, 18 and 14 in the sham, SAC and SAC+udenafil groups, respectively. All surgeries were performed under isoflurane anesthesia, and all efforts were made to minimize the suffering of the rats.

Udenafil (CAS No.; 268203-93-6, 5-[2-propoxy-5-(1-methyl-2-pyrrolidinylethylaminosulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyramidine-7-one) was obtained from Dong-A Pharmaceutical Company (Seoul, Korea). The rats in the SAC+udenafil group received daily oral administrations of udenafil (20 mg kg−1)11, 12 via gavage beginning the 3rd week after the SAC surgery until study termination, the 20th week after surgery. The rats in the other groups received vehicle (Trisol buffer solution, citrate sodium hydroxide buffer, pH 5.0, Merck, Darmstadt, Germany) treatment during the same study periods. The treatment was well tolerated by all rats. The rats were housed under standard laboratory conditions, and food and UV-sterilized tap water were provided. The study was performed in strict accordance with the recommendations of the Institutional Animal Care and Use Committee of Seoul National University Hospital (Seoul, Korea).

Creation of the SAC model

LV hypertrophy was induced by using SAC surgery (Supplementary Figure S1).13, 14 The abdominal aorta was exposed through a midline abdominal incision under sterile conditions. The abdominal aorta between the diaphragm and the renal artery was partially constricted by using a 22-gauge needle (outside diameter=0.7 mm). The needle was placed parallel to the aorta and ligated tightly to the aorta. The needle was carefully removed, leaving the vessel constricted to the diameter of the needle. This procedure produces ~50% stenosis of the aortic area.13 The rats in the sham group underwent a similar surgical procedure without aortic constriction.

Expanded methods are available in the Supplementary Material online.

Results

SAC surgery created cardiac hypertrophy

The suprarenal abdominal aortas were harvested three weeks after SAC, and the luminal cross-sectional area was measured (n=3 in each group). The mean luminal cross-sectional area of suprarenal abdominal aortas in the SAC group was significantly smaller than in the sham operation group (0.51±0.18 versus 1.26±0.37 mm2, P=0.037) (Supplementary Figure S2). There were no significant differences in body weight during the study period between the three groups (Supplementary Figure S3). Supplementary Table S1 summarizes the weights of major organs of normal rats and rats exposed to three weeks of pressure-overload. There were no significant differences in liver weights. In contrast, the rats that underwent the SAC surgery showed significantly increased heart, lung and heart/body weights and lung/body weight ratios (P<0.05).

Udenafil increased the survival of rats with cardiac hypertrophy

Five of 13 rats (38.5%) in the SAC group and two of 12 rats (16.6%) in the SAC+udenafil group died during the study period. No deaths occurred in the sham group. The first deaths occurred on the 56th and 128th days post surgery in the SAC and SAC+udenafil groups, respectively. The survival benefit of udenafil was significant based on the Kaplan–Meier survival curve (log-rank P=0.012) (Figure 1).

Figure 1
figure 1

Kaplan–Meier survival curves of the three groups. n=19, 18 and 14 in sham, SAC and SAC+UD groups, respectively. SAC, suprarenal aortic constriction; UD, udenafil.

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Udenafil increased the exercise capacity of rats with cardiac hypertrophy

The mean maximal exercise capacities (seconds to exhaustion) at baseline were not different between the three groups (P=0.982). Longitudinal changes in maximal exercise capacity are demonstrated in Figure 2. Exercise capacity was progressively impaired from the 6th week post surgery in the SAC group. Exercise capacity at the 20th week in the SAC group (447±51 s to 317±73 s, P<0.05) decreased to 70% of the exercise capacity 3 weeks post surgery. Exercise capacity was maintained in the SAC+udenafil group throughout the study period (458±107 s at the 3rd week to 448±54 s at the 20th week post surgery, P>0.05). Exercise capacity was better in the SAC+udenafil group than the SAC group at the 20th week (448±54 s for the SAC+udenafil group versus 317±73 s for the SAC group, P<0.05).

Figure 2
figure 2

Serial changes in exercise capacities of the three groups. *P<0.05 for difference from sham group; #P<0.05 for difference from SAC group. SAC, suprarenal aortic constriction; UD, udenafil.

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Udenafil improved LV systolic function and inhibited LV remodeling in rats with cardiac hypertrophy

Pre-surgery echocardiographic examinations showed no significant differences in LVSWT, LV end-diastolic dimension, LV ejection fraction (LVEF) or LV mass index between the three groups (Supplementary Table S2). Serial echocardiographic changes in LVSWT, LV end-diastolic dimension, LV mass index and LVEF in the three groups are presented in Figure 3. LVSWT increased significantly in the SAC (1.97±0.14 to 2.67±0.13 mm, P<0.05) and SAC+udenafil (2.00±0.18 to 2.78±0.15 mm, P<0.05) groups at the 3rd week post surgery compared with the baseline. The LVSWT of the SAC group increased progressively until the 9th week and slightly decreased thereafter. The LVSWT decreased consistently from the 3rd week post surgery in the SAC+udenafil group. The mean LVSWTs were 2.78±0.18 mm for the SAC and 2.38±0.13 mm for SAC+udenafil groups 20 weeks post surgery (P<0.05) (Figure 3a). LV cavity was more rapidly dilated in the SAC group than in the SAC+udenafil group. The mean LV end-diastolic dimensions were 9.84±0.59 mm for the SAC and 9.05±0.58 mm for the SAC+udenafil groups (P<0.05) at the 20th week post surgery (Figure 3b). LV mass index increased significantly in the SAC (2.84±0.42 to 3.94±0.46 g kg−1, P<0.05) and SAC+udenafil (2.75±0.32 to 3.98±0.59 g kg−1, P<0.05) groups at the 3rd week post surgery compared with baseline. LV mass index progressively increased in the SAC group throughout the study period. Udenafil treatment attenuated the increase in LV mass index (4.61±0.20 g kg−1 in the SAC group versus 3.84±0.29 g kg−1 in the SAC+udenafil group at the 20th week post surgery, P<0.05) (Figure 3c). LVEF decreased significantly in the SAC (75±4 to 61±5%, P<0.05) and SAC+udenafil (74±4 to 61±5%, P<0.05) groups at the 3rd week post surgery compared with baseline. LVEF progressively decreased throughout the study period in the SAC group, but it increased continuously in the SAC+udenafil group from the 3rd week post SAC operation. Differences in LVEF between the SAC group and the SAC+udenafil group were significant from the 12th week after SAC. The mean LVEFs were 58±3% for the SAC group and 67±3% for the SAC+udenafil group at the 20th week post surgery (P<0.05) (Figure 3d). LV fractional shortening, stroke volume and cardiac output showed similar patterns to LVEF in serial echocardiographic examinations (Supplementary Figure S4). Representative figures of echocardiographic examinations with M-mode tracings of LV are shown in Figure 3e.

Figure 3
figure 3

The results of serial echocardiographic examinations of the three groups. (a) Left ventricular septal wall thickness, (b) left ventricular end-diastolic dimension, (c) left ventricular mass index, (d) left ventricular ejection fraction, and (e) representative figures with M-mode tracings of left ventricles. *P<0.05 for difference from sham group; #P<0.05 for difference from SAC group. SAC, suprarenal aortic constriction; UD, udenafil.

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Udenafil improved LV performance as measured using invasive hemodynamic study in rats with cardiac hypertrophy

Hemodynamic studies using an LV micro-catheter were performed at the 3rd week (n=4 in each group) and the 20th week (n=7 in each group) post surgery. Mean arterial blood pressure (BP) measured at the carotid artery was higher in the SAC group at the 3rd week post surgery than in the sham control group (89.2±10.3 versus 63.0±8.2 mm Hg, P<0.05). LV end-systolic pressure (LVESP) increased in the SAC group compared with the sham control group (127±13 versus 97±11 mm Hg, P=0.05). However, LV end-diastolic pressure (LVEDP) was not different between the two groups (13.6±2.2 versus 11.4±3.2 mm Hg, P>0.05) (Figure 4a). The maximal slope of the systolic pressure increment (+dP/dt) (6756±654 versus 3648±456 mm Hg s−1, P<0.001) and the decrease in diastolic pressure (−dP/dt) (−6568±735 versus −3348±731 mm Hg s−1, P<0.001) were better in the SAC group than the sham control group (Figure 4c). Mean BPs of carotid arteries were measured at the 20th week post surgery as 73.2±12, 92.2±6.7, 85.2±10.3 mm Hg in the sham, SAC and SAC+udenafil groups, respectively. The mean carotid artery BP was higher in the SAC group than in the sham group (P<0.05), but there was no significant difference in mean carotid artery BPs between the SAC and SAC+udenafil groups (P>0.05), which suggests that udenafil is ineffective in reducing BP. Neither LVEDP nor LV end-systolic pressure were different between the three groups at the 20th week post surgery (P>0.05) (Figure 4b). The amplitudes of +dP/dt and –dP/dt signals decreased significantly in the SAC group, which indicates a decrease in cardiac contractility. However, udenafil treatment preserved these amplitudes (+dP/dt, sham versus SAC versus SAC+udenafil, 6694±1505 versus 3540±1544 versus 6518±1306 mm Hg s−1, P<0.05; -dP/dt, sham versus SAC vs. SAC+udenafil, −7306±1639 versus −3549±1790 versus −6575±1560 mm Hg s−1, P<0.05) (Figure 4d).

Figure 4
figure 4

Hemodynamic studies of left ventricles. (a) Left ventricular pressures of the sham and SAC groups at the 3rd week post surgery, and (b) left ventricular pressures of the three groups at the 20th week post surgery, (c) +dP/dt and –dP/dt of the sham and SAC groups at the 3rd week post surgery and (d) +dP/dt and –dP/dt of the three groups at the 20th week post surgery. n=4 in each group. LVEDP, left ventricular end-diastolic pressure; LVESP, left ventricular end-systolic pressure; SAC, suprarenal aortic constriction; Ud, udenafil.

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Udenafil-attenuated myocardial fibrosis, apoptosis and MMP-9 expression in rats with cardiac hypertrophy

LV wall thickness at the 3rd week post surgery increased significantly in the SAC group compared with the sham group as shown in gross examination (Supplementary Figure S5A) and hematoxylin and eosin stains (Supplementary Figure S5B). Masson’s trichrome staining revealed that the extent of interstitial and perivascular fibrosis was significantly greater in the SAC group than in the sham group at the 3rd week post surgery (Supplementary Figure S6). PDE5 expression in the myocardium of the sham group was negligible, but PDE5 expression increased in the myocardium of the rats exposed to SAC. Representative figures of PDE5 expression at the 3rd week post surgery are shown in Supplementary Figure S7.

Gross histopathological examinations at the 20th week post surgery revealed markedly increased LV wall thickness in the SAC group, but hypertrophy was attenuated in the udenafil-treated SAC group (Figure 5a). Masson’s trichrome staining revealed that the fibrotic changes in the myocardium were more prominent in the SAC group, and udenafil administration attenuated these changes at the 20th week post surgery (Figure 5b). Matrix metalloproteinase (MMP)-2 expression in the myocardium was very weak in the rats in all three groups (data not shown). MMP-9 was highly expressed in the SAC group, but udenafil treatment attenuated this expression (Figure 5c). Terminal deoxynuclotidyl transferase-mediated UTP nick-end labeling staining showed a significant increase in myocardial apoptosis in the SAC group, which was attenuated in the SAC+udenafil group (apoptotic cells per high-power field ( × 200), 3.8±2.3, 40.2±31.9 and 14.6±6.5, in sham, SAC and SAC+udenafil groups, respectively, P<0.05) (Figure 5d and Supplementary Figure S8).

Figure 5
figure 5

Histopathological examinations of left ventricles at the 20th week post surgery. (a) Gross examinations, (b) Masson’s trichrome staining showing the extent of fibrosis, (c) immunohistochemistry showing matrix metalloproteinase-9 expression and (d) TUNEL staining showing apoptosis. For TUNEL staining, n=5 in each group. SAC, suprarenal aortic constriction; TUNEL, terminal deoxynuclotidyl transferase-mediated UTP nick-end labeling; UD, udenafil.

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Udenafil augmented interleukin (IL)-10 in serum of rats with cardiac hypertrophy

Four inflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-10 and tumor necrosis factor-α, were measured in rat serum (n=4 in each group) at the 3rd and 20th weeks post surgery. IL-6 and tumor necrosis factor-α were not reliably measured because of their low concentrations. Serum IL-1β was increased in the SAC groups compared with the sham group at the 3rd week post surgery, but it did not reach statistical significance (P=0.107). Serum IL-10 levels were similar between the sham and SAC groups at the 3rd week post surgery (P=0.978). Serum IL-1β levels were not different between the three groups at the 20th week post surgery (P=0.241). However, serum IL-10 levels increased significantly in the SAC+udenafil group (94±33, 296±206, and 637±120 pg ml−1, in sham, SAC, and SAC+udenafil groups, respectively, P<0.001) (Figure 6).

Figure 6
figure 6

Serum IL-1β and IL-10 levels in rat sera. (a and b) At the 3rd week post surgery and (c and d) at the 20th week post surgery. n=4 in each group. ANOVA, analysis of variance; IL, interleukin; SAC, suprarenal aortic constriction; UD, udenafil; Veh, vehicle.

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Discussion

The study results demonstrated that the long-term use of udenafil prevented cardiac remodeling and systolic dysfunction, which improved the exercise capacity and survival in rats exposed to pressure-overload cardiac hypertrophy. Decreases in fibrosis and apoptosis of hypertrophied myocardium and MMP-9 and IL-10 modulations were proposed as potential mechanisms related to the cardio-protective effects of udenafil.

The role of PDE5 inhibition in pressure-overload cardiac hypertrophy

Cardiac hypertrophy in response to sustained pressure-overload is associated with maladaptive molecular alterations that lead to cardiac failure.15 Accumulating evidence supports the cardio-protective effects of PDE5 inhibition in pressure-overload cardiac hypertrophy. PDE5 expression increased,8, 16 and PDE5 inhibition by sildenafil suppressed LV hypertrophy and improved LV systolic function in mice exposed to chronic pressure-overload induced by thoracic aorta constriction.8 Sildenafil inhibited hypertrophic signaling pathways triggered by pressure-overload, such as phosphatidylinositol-3 kinases (PI3K)/Akt and extracellular signal-regulated kinases (ERK)1/2 pathways, which attenuated cardiac and myocyte hypertrophy and interstitial fibrosis and improved cardiac function.8 Intraperitoneal administration of sildenafil decreased cardiac hypertrophy and myocyte injury in rats in an isoproterenol-induced myocardial injury model.17 We demonstrated a marked increase in PDE5 expression in the heart of the rats subjected to SAC for 3 weeks compared with that in the hearts of the controls and a therapeutic effect of udenafil on PDE5 inhibition in this model, which is consistent with these previous reports. Our results add evidence of the important role of PDE5 in pressure-overload cardiac hypertrophy and subsequent cardiac failure, which suggests that PDE5 inhibition is a potential therapeutic target.

Effects of udenafil on IL-10 and MMP-9 modulation

To our knowledge, this is the first study to demonstrate associations of IL-10 and MMP-9 with PDE5 inhibitors in pressure-overload hypertrophy. IL-10 is a strong anti-inflammatory molecule,18 and the production of IL-10 increased during SAC-induced pro-inflammatory conditions. We demonstrated that udenafil treatment significantly augmented IL-10 levels 20 weeks after the SAC surgery, which is consistent with previous reports that improvements in cardiac function after treatment with losartan, growth hormone or immunoglobulins are associated with increased IL-10.19, 20 The increased expression and activation of MMPs were documented in failing heart, and these changes were implicated in adverse myocardial remodeling.21 The role of MMP-9 in the LV remodeling process supports our hypothesis that MMP-9 inhibition by udenafil has a role in delaying the remodeling process. The present results suggest that the cardio-protective effect of udenafil on LV remodeling and cardiac function is partially mediated via IL-10 augmentation and MMP-9 suppression.

Consideration of BP-dependent effects of udenafil

The arterial BP lowering effects of udenafil, which allowed improved LV hemodynamic performance, should also be considered to be another possible mechanism. The BP difference in carotid arteries between SAC and SAC+udenafil groups was not significant in our study, but the result could not exclude the BP lowering effects of udenafil. Arterial unloading has a significant impact on LV performance and geometry in cardiac hypertrophy.22 Therefore, the BP-dependent effect of udenafil is an important issue. Animal and clinical studies investigated the effects of udenafil on systemic arterial BP.23, 24, 25 These studies demonstrated that the impact of udenafil on systemic arterial BP was minimal and clinically insignificant, which is consistent with our finding and implies that arterial BP changes are not the main factors mediating the therapeutic potential of udenafil.

Strength of udenafil compared with sildenafil

There are major concerns regarding the side effects of PDE5 inhibitors, such as facial flushing, headache, color discrimination abnormality and cardiac events.26 Therefore, efforts are underway to develop new PDE5 inhibitors with higher potencies and a lower incidence of adverse drug reactions. Udenafil has a number of strengths compared with sildenafil. Udenafil is comparable to sildenafil in PDE5 selectivity,9 and it exhibits relatively few side effects and a longer half-life than sildenafil.27 Udenafil can also be administered without limitations of meal conditions, and modifications of the dosing regimen are unnecessary.10 Many previous trials demonstrated that long-term sildenafil treatment improved exercise capacity in patients with heart failure or pulmonary hypertension.28, 29 Udenafil has a longer half-life and action of duration than sildenafil, and once-daily dosing may be sufficient to obtain favorable effects.30 Our results suggest that udenafil plays an important role in the treatment of hypertrophic heart disease with practical advantages over sildenafil.

Clinical implications

Pathological cardiac hypertrophy is considered an irreversible pathway. However, the traditional view of the irreversibility of pathological cardiac hypertrophy was challenged recently. Several studies reported that certain devices or drugs can reverse cardiac hypertrophy, prevent the remodeling process and improve cardiac contractibility of failing hearts.8, 31, 32 The regression of pathological cardiac hypertrophy has become a target of cardiac medicine based on these findings.33, 34 The exploration of molecular targets that are involved in the regression of pathological cardiac hypertrophy would lead to alternative therapeutic options for heart failure. However, no remarkable progress in the development of new therapeutics targeting cardiac hypertrophy has occurred, despite this effort. The results of this study have clinical implications and suggest udenafil to be another therapeutic option for the regression of established pathological cardiac hypertrophy. Our group is performing clinical trials on the therapeutic effect of udenafil in patients with chronic systolic heart failure.35

Study limitations

This study has several limitations. First, the study groups were not blinded to the researchers. Second, udenafil demonstrated several changes in this study, but the mechanisms mediating the action of udenafil are largely unknown. Third, there were no comparative experiments using sildenafil. Lastly, direct application of our results to humans may be difficult because much higher dosages of udenafil were used in this study than those found in clinical use.

Conclusion

Long-term udenafil use prevents cardiac remodeling, which improves exercise capacity and survival in rats exposed to pressure-overload cardiac hypertrophy. Decreases in fibrosis and apoptosis of the hypertrophied myocardium and modulation of MMP and inflammatory cytokines are proposed as potential mechanisms related to the cardio-protective effects of udenafil.