**3. PDE-5 Inhibitors protect against doxorubicin-induced cardiac dysfunction**

A number of pioneering investigations from our laboratory have demonstrated that PDE-5 inhibitors attenuate doxorubicin-induced cardiomyopathy in animal and cell models (Koka et al., 2010; Das et al., 2010). In a recent study, we tested whether sildenafil potentiates the antitumor efficacy of doxorubicin in prostate cancer. Our results show that doxorubicin and sildenafil induce a potent antitumor effect in prostate cancer while simultaneously providing a cardioprotective effect. This study was an elegant sequel to our previous work demonstrating that sildenafil attenuated doxorubicin-induced cardiomyopathy in mice (Fisher et al., 2005). In that study, we showed that treatment with sildenafil attenuates the decline in LV developed pressure caused by doxorubicin treatment. An important question, however, was whether sildenafil interferes with the anti-tumor effect of doxorubicin. Our recent study showed that sildenafil ameliorates doxorubicin-induced cardiac dysfunction without interfering with its chemotherapeutic benefits (Das et al., 2010). Cardiac function in nude mice with tumor xenografts was monitored by Doppler echocardiography using the Vevo770 imaging system (VisualSonics, Toronto, Canada) as previously reported (Salloum et al., 2008a). A slight increase in LVEDD and LVESD were observed with doxorubicin. LVFS and LVEF declined in doxorubicin-treated mice. Sildenafil co-treatment with doxorubicin improved LVFS and LVEF compared with the doxorubicin-treated group (P < 0.05). No differences in heart rate were observed between control, doxorubicin, and doxorubicin and sildenafil groups. Sildenafil-treated animals showed lower heart rates compared with other groups (P < 0.01; n = 8). These data suggest that changes in LVFS or LVEF were independent of heart rate.

In a separate study, tadalafil improved left ventricular function and prevented cardiomyocyte apoptosis in doxorubicin-induced cardiomyopathy through mechanisms involving upregulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of doxorubicin (Koka et al., 2010). In these studies, adult male CF-1 mice were randomized to receive saline (0.2 ml i.p.), doxorubicin (15 mg/kg i.p.), or doxorubicin + tadalafil (4 mg/kg p.o. daily) for 9 days starting 3 days before doxorubicin treatment. We chose to use a single dose of doxorubicin at 15 mg/kg i.p., which has been reported to be cardiotoxic. LV function was significantly impaired 5 days after doxorubicin treatment. However, mice treated with doxorubicin + tadalafil showed preserved fractional shortening and ejection fraction compared with those treated with doxorubicin as shown in Figure 6 (n = 6, p < 0.05). In addition, the LV systolic pressure decreased 36%, +dp/dtmax decreased 63%, −dp/dtmax decreased 57%, and heart rate decreased 30% as compared with the controls (P < 0.05). In contrast, mice treated with doxorubicin + tadalafil showed improved LV function (i.e., LV systolic pressure, 33%; +dp/dtmax, 35%; −dp/dtmax, 46%, and heart rate, 27%) as compared with the group treated with doxorubicin alone (n = 6, p < 0.05).

(3.5±0.1 mm and 2.4±0.2 mm, respectively). In contrast, no dilatation was detected in sildenafil (3.0±0. mm and 1.4±0.1 mm, respectively) and vardenafil (2.9±0.3 mm and 1.4±0.2 mm, respectively) groups. Fractional shortening decreased at 7 days post infarction with saline (30±4%; P<0.05), but was preserved with sildenafil (52±2%) and vardenafil (53±5%). These data clearly suggest that PDE-5 inhibitors induce powerful cardioprotection in female mice as well. For this reason, PDE-5 inhibition may be a novel therapeutic strategy against

**3. PDE-5 Inhibitors protect against doxorubicin-induced cardiac dysfunction**  A number of pioneering investigations from our laboratory have demonstrated that PDE-5 inhibitors attenuate doxorubicin-induced cardiomyopathy in animal and cell models (Koka et al., 2010; Das et al., 2010). In a recent study, we tested whether sildenafil potentiates the antitumor efficacy of doxorubicin in prostate cancer. Our results show that doxorubicin and sildenafil induce a potent antitumor effect in prostate cancer while simultaneously providing a cardioprotective effect. This study was an elegant sequel to our previous work demonstrating that sildenafil attenuated doxorubicin-induced cardiomyopathy in mice (Fisher et al., 2005). In that study, we showed that treatment with sildenafil attenuates the decline in LV developed pressure caused by doxorubicin treatment. An important question, however, was whether sildenafil interferes with the anti-tumor effect of doxorubicin. Our recent study showed that sildenafil ameliorates doxorubicin-induced cardiac dysfunction without interfering with its chemotherapeutic benefits (Das et al., 2010). Cardiac function in nude mice with tumor xenografts was monitored by Doppler echocardiography using the Vevo770 imaging system (VisualSonics, Toronto, Canada) as previously reported (Salloum et al., 2008a). A slight increase in LVEDD and LVESD were observed with doxorubicin. LVFS and LVEF declined in doxorubicin-treated mice. Sildenafil co-treatment with doxorubicin improved LVFS and LVEF compared with the doxorubicin-treated group (P < 0.05). No differences in heart rate were observed between control, doxorubicin, and doxorubicin and sildenafil groups. Sildenafil-treated animals showed lower heart rates compared with other groups (P < 0.01; n = 8). These data suggest that changes in LVFS or

In a separate study, tadalafil improved left ventricular function and prevented cardiomyocyte apoptosis in doxorubicin-induced cardiomyopathy through mechanisms involving upregulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of doxorubicin (Koka et al., 2010). In these studies, adult male CF-1 mice were randomized to receive saline (0.2 ml i.p.), doxorubicin (15 mg/kg i.p.), or doxorubicin + tadalafil (4 mg/kg p.o. daily) for 9 days starting 3 days before doxorubicin treatment. We chose to use a single dose of doxorubicin at 15 mg/kg i.p., which has been reported to be cardiotoxic. LV function was significantly impaired 5 days after doxorubicin treatment. However, mice treated with doxorubicin + tadalafil showed preserved fractional shortening and ejection fraction compared with those treated with doxorubicin as shown in Figure 6 (n = 6, p < 0.05). In addition, the LV systolic pressure decreased 36%, +dp/dtmax decreased 63%, −dp/dtmax decreased 57%, and heart rate decreased 30% as compared with the controls (P < 0.05). In contrast, mice treated with doxorubicin + tadalafil showed improved LV function (i.e., LV systolic pressure, 33%; +dp/dtmax, 35%; −dp/dtmax, 46%, and heart

rate, 27%) as compared with the group treated with doxorubicin alone (n = 6, p < 0.05).

ischemia/reperfusion injury in women with coronary artery disease.

LVEF were independent of heart rate.

Fig. 6. Tadalafil attenuates doxorubicin-induced LV dysfunction

#### **4. PDE-5 inhibitors protect against hypertrophy-induced cardiac dysfunction**

Sustained pressure overload leads to cellular and molecular changes that are initially activated as compensatory mechanisms but later become maladaptive and contribute to progressive cardiac dysfunction and heart failure. This response involves a combination of complex signaling and transcription pathways that induce hypertrophic remodeling (Frey & Olson, 2003; Frey et al., 2004). The heart appears to have an intrinsic signaling system coupled to cGMP that can inhibit myocardial proliferative responses. Several studies using approaches that involve enhanced cGMP synthesis or prevention of its degradation have

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 107

mortem analysis confirmed that both heart and lung weights, normalized to tibia length, were lower with sildenafil treatment. Moreover, cardiomyocyte cross-sectional dimension and interstitial and perivascular fibrosis was also reduced in sildenafil-treated myocardium.

Duchenne muscular dystrophy (DMD) is a degenerative, muscle-wasting disease caused by mutations in the dystrophin gene. The total loss of dystrophin mainly affects skeletal muscle and results in impaired respiratory function, primarily in older boys (Finsterer & Stöllberger, 2003; Adamo, 2010). Due to remarkable improvement of noninvasive respiratory support in the recent past, the lifespan of patients with DMD has increased. Unfortunately, this was also associated with an increase in the incidence of complications and eventual mortality from cardiomyopathy (McNally, 2008). Cardiomyopathy is a delayed symptom of the disease that usually develops by the second decade of life, with more than 90% of patients presenting clinical symptoms by 18 y of age (Finsterer & Stöllberger, 2003). Loss of cardiac dystrophin eventually leads to dilated cardiomyopathy, which manifests as congestive heart failure in at least 20% of patients (Finsterer & Stöllberger, 2003). Current treatment options for heart failure associated with DMD include angiotensin converting enzyme inhibitors and β-blockers. Despite the moderate benefits provided by these medications in patients with systolic heart failure, similar advantages have not been observed in dystrophic patients with features of systolic and diastolic dysfunction (Bushby, 2003). These findings highlight the need for treatments that slow the development of cardiomyopathy in DMD and improve cardiac function in older patients

It has been shown that stimulation of cGMP synthesis by overexpression of cardiac-specific neuronal (n)NOS reduces impulse-conduction defects in dystrophin-deficient (mdx) mice (Wehling-Henricks et al., 2005; Wehling et al., 2001). Similarly, increased particulate guanylyl cyclase activity in young mdx mice has also been shown to decrease susceptibility to cardiac damage during sympathetic stress (Khairallah et al., 2008). These findings clearly implicate reduced NO-cGMP signaling as a key contributor to myocardial pathogenesis in patients with DMD. Therefore, it is plausible that restoration of NO signaling, particularly by preservation of cGMP, may provide therapeutic benefit to dystrophic hearts. In a recent study, Adamo et al. tested whether chronic inhibition of PDE-5 with sildenafil would

reverse cardiac dysfunction in the mdx mouse model of DMD (Adamo et al., 2010)

Chronic sildenafil treatment prevented LV functional deficits in aging mdx mice. Furthermore, late sildenafil treatment, i.e. after developing cardiomyopathy, reversed the

Conventional echocardiography and tissue Doppler analysis were used to monitor the development of LV dysfunction in aging mdx mice. Both the myocardial performance index (MPI) and ratios of early diastolic velocity (Ea) to peak velocity with atrial contraction (Aa) were calculated. MPI is a sensitive measure of left ventricular systolic and diastolic performance, whereas the Ea/Aa largely reflects diastolic function. The majority of patients with DMD exhibit diastolic dysfunction and impaired myocardial performance, which can be identified by increased MPI (Bahler et al., 2005). This dysfunction usually precedes the

**5. PDE-5 inhibitors reverse cardiac dysfunction in Duchenne muscular** 

**dystrophy** 

with established cardiomyopathy.

established symptoms.

been shown to blunt hypertrophy despite sustained pressure overload or neurohormonal stress. Interestingly, although cGMP synthesis is often increased by chronic exposure to such stresses, this increase appears to be ineffective to impede hypertrophy and remodeling progression, likely due to increase in PDE-5 expression and activity that accompany such stressors. For this reason, the use of PDE-5 inhibitors to reduce the catabolism may augment cGMP-dependent antihypertrophic effects. In the study by Takimoto et al., the authors show that PDE-5 inhibition with sildenafil prevents cardiac chamber, cellular and molecular remodeling induced by pressure overload (Takimoto et al., 2005). They next tested a more clinically relevant question of whether inhibition of PDE-5 can reverse pre-existing hypertrophy. Mice were exposed to transverse aortic constriction for 7–10 days, which increased heart mass by 63% (P < 0.005) without chamber dilatation. After hypertrophy was established, these mice were divided into 2 groups that received either sildenafil or vehicle for an additional 2 weeks. Cardiomyocyte hypertrophy and interstitial fibrosis were observed in mice exposed to 1 week of transverse aortic constriction, and both reversed to baseline with sildenafil treatment. Serial echocardiography also showed a gradual decline in LV mass and wall thickness, with preservation of systolic ejection in sildenafil-treated mice (Figure 7).

Fig. 7. Sildenafil attenuates and reverses hypertrophy-induced cardiac dysfunction

Another study by Nagayama et al. showed that delayed sildenafil treatment suppresses progressive cardiac dilatation, dysfunction, fibrosis, and hypertrophy in hearts subjected to sustained pressure-overload (Nagayama et al., 2009). In this study, following 3-week transverse aortic constriction, hearts had a +135% increase in LV mass, chamber end-systolic (+91%) and end-diastolic (+10%) dimensions, and reduced fractional shortening (−42%). Subsequent treatment with sildenafil fully arrested progressive remodeling, whereas control hearts further dilated and hypertrophied after 9-week transverse aortic constriction. Post-

been shown to blunt hypertrophy despite sustained pressure overload or neurohormonal stress. Interestingly, although cGMP synthesis is often increased by chronic exposure to such stresses, this increase appears to be ineffective to impede hypertrophy and remodeling progression, likely due to increase in PDE-5 expression and activity that accompany such stressors. For this reason, the use of PDE-5 inhibitors to reduce the catabolism may augment cGMP-dependent antihypertrophic effects. In the study by Takimoto et al., the authors show that PDE-5 inhibition with sildenafil prevents cardiac chamber, cellular and molecular remodeling induced by pressure overload (Takimoto et al., 2005). They next tested a more clinically relevant question of whether inhibition of PDE-5 can reverse pre-existing hypertrophy. Mice were exposed to transverse aortic constriction for 7–10 days, which increased heart mass by 63% (P < 0.005) without chamber dilatation. After hypertrophy was established, these mice were divided into 2 groups that received either sildenafil or vehicle for an additional 2 weeks. Cardiomyocyte hypertrophy and interstitial fibrosis were observed in mice exposed to 1 week of transverse aortic constriction, and both reversed to baseline with sildenafil treatment. Serial echocardiography also showed a gradual decline in LV mass and wall thickness, with preservation of systolic ejection in sildenafil-treated mice (Figure 7).

Fig. 7. Sildenafil attenuates and reverses hypertrophy-induced cardiac dysfunction

Another study by Nagayama et al. showed that delayed sildenafil treatment suppresses progressive cardiac dilatation, dysfunction, fibrosis, and hypertrophy in hearts subjected to sustained pressure-overload (Nagayama et al., 2009). In this study, following 3-week transverse aortic constriction, hearts had a +135% increase in LV mass, chamber end-systolic (+91%) and end-diastolic (+10%) dimensions, and reduced fractional shortening (−42%). Subsequent treatment with sildenafil fully arrested progressive remodeling, whereas control hearts further dilated and hypertrophied after 9-week transverse aortic constriction. Postmortem analysis confirmed that both heart and lung weights, normalized to tibia length, were lower with sildenafil treatment. Moreover, cardiomyocyte cross-sectional dimension and interstitial and perivascular fibrosis was also reduced in sildenafil-treated myocardium.
