**2.1 Sildenafil attenuates left ventricular dysfunction in ischemic heart failure**

Salloum et al. showed that chronic treatment with sildenafil preserves cardiomyocytes post AMI through reduction of myocardial necrosis, apoptosis and hypertrophy thereby limiting the progression of HF (Salloum et al., 2008a). This study used a murine model of post-MI remodeling by permanent ligation of the left coronary artery. The experimental protocol is illustrated in Figure 1. LV function was assessed at 7 and 28 days post MI. Cardiac function

Fig. 1. Experimental protocol illustrating various parameters studied at different time points

suboptimal due to endothelial dysfunction [damaged NO synthase (NOS)], and recently myocardial PDE-5 expression has been shown to increase in patients with heart failure (Pokreisz et al., 2009). In this regard, targeting PDE-5 is a promising therapeutic approach

A number of pioneering investigations from our laboratory have demonstrated that PDE-5 inhibitors attenuate ischemic injury in animal and cell models (Ockaili et al., 2002; Salloum et al., 2003). In animal models, sildenafil and vardenafil exerted an infarct-sparing effect when given before ischemia (Ockaili et al., 2002; Salloum et al., 2003; Salloum et al., 2006) or at the time of reperfusion (Salloum et al., 2007). Furthermore, chronic treatment with sildenafil immediately after permanent occlusion of the left descending coronary artery (LAD) in mice attenuated ischemic cardiomyopathy (Salloum et al., 2008a). These cardioprotective effects are mediated by activation of protein kinase G (PKG), increased expression of endothelial and inducible nitric oxide synthase (eNOS & iNOS), and augmented Bcl-2/Bax ratio. Due to their powerful anti-ischemic effects, PDE-5 inhibitors became promising candidates for the preservation of cardiac function following AMI. In fact, several studies demonstrated that PDE-5 inhibition preserved left ventricular (LV)

**2. PDE-5 Inhibitors preserve myocardial function following infarction** 

**2.1 Sildenafil attenuates left ventricular dysfunction in ischemic heart failure** 

Salloum et al. showed that chronic treatment with sildenafil preserves cardiomyocytes post AMI through reduction of myocardial necrosis, apoptosis and hypertrophy thereby limiting the progression of HF (Salloum et al., 2008a). This study used a murine model of post-MI remodeling by permanent ligation of the left coronary artery. The experimental protocol is illustrated in Figure 1. LV function was assessed at 7 and 28 days post MI. Cardiac function

> **Sildenafil (0.71 mg/kg; ip; BID) or Saline (volume matched; ip; BID)**

**Week 1 Week 2 Week 3 Week 4**

**Daily monitoring: Pain, appetite/drinking, behavior and responsiveness**

Fig. 1. Experimental protocol illustrating various parameters studied at different time points

•**Echocardiography** •**Body Weight** •**Heart weight** •**Lung weight** •**TUNEL** •**Survival**

for treatment of cardiovascular disease and dysfunction.

function in failing hearts as discussed in the following sections.

•**Echocardiography** •**Body Weight** •**Heart weight** •**Lung weight** •**TUNEL** •**Survival**

**LAD Occlusion or Sham Surgery**

**Baseline Echo**

**8 week-old ICR Mice**

> **24 hr Infarct Size**

was evaluated by echocardiography using the Vevo770TM imaging system (VisualSonics, Inc., Toronto, Canada). A 30-MHz probe was utilized to obtain two-dimensional, M-mode and Doppler imaging from parasternal short-axis view at the level of the papillary muscles and the apical four-chamber view (Schiller et al., 1989). M-mode images of the LV were obtained and systolic and diastolic wall thickness (anterior and posterior) and LV endsystolic and end-diastolic diameters (LVESD and LVEDD, respectively) were measured.

Figure 2 is representative of M-mode images from mice on day 28 post MI. The hearts from sham and sildenafil-treated mice exhibited a smaller LV cavity and thicker infarct wall compared to the saline-treated mice. Increase in LVEDD, LVESD and a decrease in anterior wall diastolic thickness (AWDT), anterior wall systolic thickness (AWST) and fractional shortening (FS) in saline- and sildenafil-treated mice (vs. baseline and sham) were observed on day 7 and 28.

Fig. 2. M-mode images from mouse LV treated with sildenafil or vehicle 28 days after MI

Sildenafil-treated mice had smaller LVEDD, LVESD, greater FS, and lower Tei index (reflecting better myocardial performance) on day 7 and 28 as compared to saline-treated group (P<0.05, Figure 3). Sildenafil-treated animals also had a shorter isovolumetric relaxation time (reflective of lower LV end-diastolic pressure) 28 days after AMI when compared to saline-treated animals (11±3 vs. 27±7 ms, respectively, P=0.03), which was not different from sham operated animals (10±3, P=NS). AWDT and AWST were also greater in sildenafil-treated animals (vs. saline-treated animals, P<0.05) on day 7 and 28 post MI showing a protective effect in the peri-infarct region, while no differences in PWDT and PWST were seen. Aneurysmatic dilatation of the anterior wall and apex was observed on day 28 in 90% of saline-treated mice and 62% of sildenafil treated animals (P>0.05).

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 101

Fig. 4. Tadalafil preserves LV function at 24 hours following MI through PKG and H2S

**2.3 Sildenafil treatment 3 days following MI mitigates the progression of heart failure**  In the previous studies, PDE-5 inhibitors were administered either shortly prior to or immediately after infarction. This raises the question whether the preservation of cardiac function observed was a true phenomenon, or it was simply secondary to the anti-infarct effect of these drugs. Specially, little is known about the effects of PDE-5 inhibition on limiting adverse remodeling independent of its ability to modulate infarct size. This concept is clinically relevant, particularly in patients with advanced ischemic HF, because necrosis

signaling

Moreover, the number of aneurysmatic segments [based on a 16-segment map (Schiller et al., 1989)] was 2.9 in saline-treated animals vs. 1.1 in sildenafil-treated animals (P<0.05).

Fig. 3. Echocardiography results of LV function at 28 days post-MI in sildenafil- and salinetreated mice

#### **2.2 Tadalafil preserves left ventricular function following MI through PKG-dependent generation of hydrogen sulfide**

We also studied the effect of a longer acting PDE-5 inhibitor, tadalafil, on cardiac function in an acute model of myocardial infarction (Salloum et al., 2009). After baseline transthoracic echocardiography, adult male mice were injected i.p. with vehicle (10% DMSO) or tadalafil (1 mg/kg) with or without KT5823 (KT, PKG blocker, 1 mg/kg) or dl-propargylglycine [PAG, Cystathionine-γ-lyase (CSE, H2S-producing enzyme) blocker; 50 mg/kg] 1 h prior to coronary artery ligation for 30 min and reperfusion for 24 h, whereas C57BL-wild type and CSEknockout mice were treated with either vehicle or tadalafil. After reperfusion, repeat echocardiography was performed. Similar to sildenafil, tadalafil preserved cardiac performance following MI as compared to vehicle. In this study, since ischemia was limited to 30 minutes, none of the groups presented with significant LV dilatation at 24 h post infarction, however, tadalafil preserved fractional shortening (FS: 31±1.5%) compared to control (FS: 22±4.8%, P<0.05, Figure 4). Baseline FS was 44±1.7%. KT and PAG abrogated the preservation of LV function with tadalafil by a decline in FS to 17±1% and 23±3%, respectively.

Moreover, the number of aneurysmatic segments [based on a 16-segment map (Schiller et al., 1989)] was 2.9 in saline-treated animals vs. 1.1 in sildenafil-treated animals (P<0.05).

**AWDT (mm)**

**A**

**0.00 0.15 0.30 0.45 0.60 0.75 0.90**

**0.25 0.50 0.75 1.00 1.25 1.50**

**Tei**

**Saline (N=6) Sildenafil (N=6) Sham (N=6)**

Fig. 3. Echocardiography results of LV function at 28 days post-MI in sildenafil- and saline-

**2.2 Tadalafil preserves left ventricular function following MI through PKG-dependent** 

We also studied the effect of a longer acting PDE-5 inhibitor, tadalafil, on cardiac function in an acute model of myocardial infarction (Salloum et al., 2009). After baseline transthoracic echocardiography, adult male mice were injected i.p. with vehicle (10% DMSO) or tadalafil (1 mg/kg) with or without KT5823 (KT, PKG blocker, 1 mg/kg) or dl-propargylglycine [PAG, Cystathionine-γ-lyase (CSE, H2S-producing enzyme) blocker; 50 mg/kg] 1 h prior to coronary artery ligation for 30 min and reperfusion for 24 h, whereas C57BL-wild type and CSEknockout mice were treated with either vehicle or tadalafil. After reperfusion, repeat echocardiography was performed. Similar to sildenafil, tadalafil preserved cardiac performance following MI as compared to vehicle. In this study, since ischemia was limited to 30 minutes, none of the groups presented with significant LV dilatation at 24 h post infarction, however, tadalafil preserved fractional shortening (FS: 31±1.5%) compared to control (FS: 22±4.8%, P<0.05, Figure 4). Baseline FS was 44±1.7%. KT and PAG abrogated the preservation

of LV function with tadalafil by a decline in FS to 17±1% and 23±3%, respectively.

**0.1 0.2 0.3 0.4 0.5 0.6**

**index**

**C**

**B**

**AWST (mm)**

**baseline 7 days 28 days sham**

**P=0.21 P=0.017**

**P=0.14 P=0.001**

**P=0.019 P=0.032**

**D**

**F**

**E**

**0.0 baseline 7 days 28 days sham**

**0.00 baseline 7 days 28 dayssham**

**baseline 7 days 28 days sham**

**P=0.003 P<0.001**

**P=0.011 P=0.007**

**P<0.001**

**0.0 baseline 7 days 28 days sham**

**0.0 baseline 7 days 28 days sham**

**P=0.003**

**LVEDD (mm)**

**0.0 1.0 2.0 3.0 4.0 5.0 6.0**

**1.0 2.0 3.0 4.0 5.0 6.0**

**FS (%)**

treated mice

**LVESD (mm)**

**10.0 20.0 30.0 40.0 50.0 60.0**

**generation of hydrogen sulfide** 

Fig. 4. Tadalafil preserves LV function at 24 hours following MI through PKG and H2S signaling

#### **2.3 Sildenafil treatment 3 days following MI mitigates the progression of heart failure**

In the previous studies, PDE-5 inhibitors were administered either shortly prior to or immediately after infarction. This raises the question whether the preservation of cardiac function observed was a true phenomenon, or it was simply secondary to the anti-infarct effect of these drugs. Specially, little is known about the effects of PDE-5 inhibition on limiting adverse remodeling independent of its ability to modulate infarct size. This concept is clinically relevant, particularly in patients with advanced ischemic HF, because necrosis

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 103

protective effects against ischemia/reperfusion injury in the infant rabbit hearts as well. In this study, we used the model of coronary artery occlusion and reperfusion in infant rabbits (Bremer et al., 2005), which is similar to our previously described adult rabbit model of myocardial infarction. The benefits of this work are immense since this model may be applicable in pediatrics, and especially in pediatric cardiovascular surgery where there may be periods of ischemia/reperfusion injury. Also, we used two-dimensional (2D) and Doppler trans-esophageal echocardiography (TEE) for the estimation of LV cardiac output (LVCO) and aortic velocity time integral (VTI) in this model. A 10-Fr AcuNav diagnostic ultrasound probe (Acuson Corp., Siemens, Iselin, NJ) was inserted into the esophagus, at baseline, after the 30 min period of ischemia, and after 3 h of reperfusion in both the control and sildenafil groups. Standardized 2D imaging in a long axis view of the LV to show LV inflow across the mitral valve and LV outflow tract (LVOT) was obtained. Aortic flow Doppler across the aortic valve was performed in a long axis view of the LV and LVOT to obtain LVCO. The standard equation: mean velocity (cm/s) × flow area (cm2) × 60 (s/min), where mean velocity (cm/s) = VTI (in cm/beat) ÷ RR interval (s/beat), was used to obtain LVCO, expressed in milliliters per minute. Laminar Doppler flow across the aortic valve confirmed the absence of aortic stenosis. Color Doppler assessment was made of both the aortic and mitral valves again in the long axis view at baseline, after the ischemic period, and after 3 h of reperfusion for the presence or absence of mitral or aortic regurgitation. Subjective functional assessment was also made after ischemia and reperfusion to demonstrate at least left ventricular apical diminished contractility to confirm infarction. In this study, we showed that both the control and sildenafil-treated groups had comparable LVCO and aortic VTI at baseline. The controls had a decline in LVCO and aortic VTI immediately after the 30-min period of ischemia (28% and 27% lower than baseline values, respectively, p < 0.05), whereas the LVCO and aortic VTI increased in the sildenafil group after ischemia (43% and 45% higher than baseline values, respectively, n = 6 per group, p < 0.05). Both groups, however, had significant decline in LVCO after 3 h of reperfusion (54% of baseline in the sildenafil group, p < 0.05, and 62% of baseline in the control group, p < 0.05), and were not statistically significantly different from each other (n = 4–6 per group). Both groups demonstrated a decrease in aortic VTI after 3 h of reperfusion. However, this decline was only statistically significant in the control group compared with baseline values. None of the rabbits had aortic stenosis or developed aortic regurgitation for the duration of the study. Moreover, both the control and sildenafil groups demonstrated a comparable amount of mitral regurgitation (no more than mild) after ischemia/reperfusion, and none of

the rabbits had baseline mitral regurgitation.

**2.5 Sildenafil and vardenafil preserve LV function in female mice** 

Since the impact of PDE-5 inhibitors on the female cardiovascular system following ischemia remains unknown, we interrogated the effect of sildenafil and vardenafil on ischemia/reperfusion injury in female mice. In this study, adult female mice were pretreated (ip, bid) with sildenafil (0.7 mg/kg), vardenafil (0.14 mg/kg) or saline one hour before left coronary artery ligation for 30 minutes and reperfusion for 24 hours (Salloum et al., 2008b). Cardiac function, evaluated using echocardiography, showed that LV enddiastolic and end-systolic diameters increased 7 days post myocardial infarction with saline

has a negligible role in a post-infarct setting (Anversa et al., 1993). To address this question, we administered sildenafil 3 days following MI (Chau et al., 2011). Specifically, we sought to determine if sildenafil treatment following LV dysfunction, defined as FS less than 25% at day 3 post-MI, could prevent the progression of HF in a permanent LAD occlusion model. At 3 days post MI, mice receiving sildenafil or saline (control) treatment had similar FS (18±1% and 19±1%, respectively, P>0.05) as compared to baseline value of 47±1%. At days 7 and 28 post-MI, sildenafil-treated group had a significantly higher FS than saline-treated mice (P<0.05). Both LVEDD and LVESD were increased in saline-treated mice as compared to sildenafil-treated mice (P<0.05), indicating more dilatation. Moreover, AWDT was greater in sildenafil-treated animals versus saline-treated animals (P<0.05) on day 28 post-MI. Fractional shortening of sham-operated mice was 43±1.0% at 28 days post left thoracotomy. An increase in LVEDD from a baseline value of 3.5±0.1 mm and a decrease in FS in salineand sildenafil-treated mice as compared to baseline and sham-operated mice (P<0.05) was observed on days 3 and 28, as shown in figure 5.

Fig. 5. Late sildenafil treatment preserves LV function and attenuates the progression of ischemic heart failure

#### **2.4 Sildenafil preserves LV function in infant rabbits**

Although we previously showed that PDE-5 inhibitors induce powerful preconditioninglike protective effects in the ischemic heart, it is not known whether sildenafil exerts similar

has a negligible role in a post-infarct setting (Anversa et al., 1993). To address this question, we administered sildenafil 3 days following MI (Chau et al., 2011). Specifically, we sought to determine if sildenafil treatment following LV dysfunction, defined as FS less than 25% at day 3 post-MI, could prevent the progression of HF in a permanent LAD occlusion model. At 3 days post MI, mice receiving sildenafil or saline (control) treatment had similar FS (18±1% and 19±1%, respectively, P>0.05) as compared to baseline value of 47±1%. At days 7 and 28 post-MI, sildenafil-treated group had a significantly higher FS than saline-treated mice (P<0.05). Both LVEDD and LVESD were increased in saline-treated mice as compared to sildenafil-treated mice (P<0.05), indicating more dilatation. Moreover, AWDT was greater in sildenafil-treated animals versus saline-treated animals (P<0.05) on day 28 post-MI. Fractional shortening of sham-operated mice was 43±1.0% at 28 days post left thoracotomy. An increase in LVEDD from a baseline value of 3.5±0.1 mm and a decrease in FS in salineand sildenafil-treated mice as compared to baseline and sham-operated mice (P<0.05) was

observed on days 3 and 28, as shown in figure 5.

**3 d MI 2 8d Sham 28 d MI +** 

**\* P<0.05 vs. Saline at 7d # P<0.05 vs. Salin e at 28d ( n=10-12/group )**

**<sup>3</sup> <sup>7</sup> <sup>28</sup> 0.0**

**Days post MI**

**Sa line Silde na fil**

**\***

**3 4 7 28**

**Day s pos t MI**

**\* P< 0. 05 vs. Salin e a t 28d ( n =1 0-1 2/grou p )**

**0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9**

ischemic heart failure

**LV Ejection Fraction**

**( %)**

**AWDT ( mm)**

**A. B.**

**Sali ne**

**F. G.**

**# #**

**\***

**\***

**2.4 Sildenafil preserves LV function in infant rabbits** 

**Fasu d il**

**Saline**

**Sildenafil Fasudil**

**C. D. E.**

**28 d MI + Sild enafil**

> **3.5 4.0 4.5 5.0 5.5 6.0**

**LV End Diast olic Diameter**

**( mm)** **28 d MI + Fasud il**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group )**

**3 4 7 28**

**3 4 7 28**

**Days post MI**

**3.0 3.5 4.0 4.5 5.0 5.5**

**LV End Systolic Diamet**

**(mm)**

 **er**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group)**

**\***

**Saline**

**# Sildenafil**

**# Fasudil**

**LV Fract ional Sho rtening**

**(%)**

**Days post MI**

**Saline Sildenafil (n = 10-12/group)**

**3 4 7 28**

**Days post MI \***

**Saline**

**Saline**

**Sildenafil**

**# # Fasudil**

**# Fasudil**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group)**

**Sildenafil**

**#**

**Fasudil**

**Heart Rate (bpm)**

Fig. 5. Late sildenafil treatment preserves LV function and attenuates the progression of

Although we previously showed that PDE-5 inhibitors induce powerful preconditioninglike protective effects in the ischemic heart, it is not known whether sildenafil exerts similar

**3 4 7 2 8**

**\***

protective effects against ischemia/reperfusion injury in the infant rabbit hearts as well. In this study, we used the model of coronary artery occlusion and reperfusion in infant rabbits (Bremer et al., 2005), which is similar to our previously described adult rabbit model of myocardial infarction. The benefits of this work are immense since this model may be applicable in pediatrics, and especially in pediatric cardiovascular surgery where there may be periods of ischemia/reperfusion injury. Also, we used two-dimensional (2D) and Doppler trans-esophageal echocardiography (TEE) for the estimation of LV cardiac output (LVCO) and aortic velocity time integral (VTI) in this model. A 10-Fr AcuNav diagnostic ultrasound probe (Acuson Corp., Siemens, Iselin, NJ) was inserted into the esophagus, at baseline, after the 30 min period of ischemia, and after 3 h of reperfusion in both the control and sildenafil groups. Standardized 2D imaging in a long axis view of the LV to show LV inflow across the mitral valve and LV outflow tract (LVOT) was obtained. Aortic flow Doppler across the aortic valve was performed in a long axis view of the LV and LVOT to obtain LVCO. The standard equation: mean velocity (cm/s) × flow area (cm2) × 60 (s/min), where mean velocity (cm/s) = VTI (in cm/beat) ÷ RR interval (s/beat), was used to obtain LVCO, expressed in milliliters per minute. Laminar Doppler flow across the aortic valve confirmed the absence of aortic stenosis. Color Doppler assessment was made of both the aortic and mitral valves again in the long axis view at baseline, after the ischemic period, and after 3 h of reperfusion for the presence or absence of mitral or aortic regurgitation. Subjective functional assessment was also made after ischemia and reperfusion to demonstrate at least left ventricular apical diminished contractility to confirm infarction. In this study, we showed that both the control and sildenafil-treated groups had comparable LVCO and aortic VTI at baseline. The controls had a decline in LVCO and aortic VTI immediately after the 30-min period of ischemia (28% and 27% lower than baseline values, respectively, p < 0.05), whereas the LVCO and aortic VTI increased in the sildenafil group after ischemia (43% and 45% higher than baseline values, respectively, n = 6 per group, p < 0.05). Both groups, however, had significant decline in LVCO after 3 h of reperfusion (54% of baseline in the sildenafil group, p < 0.05, and 62% of baseline in the control group, p < 0.05), and were not statistically significantly different from each other (n = 4–6 per group). Both groups demonstrated a decrease in aortic VTI after 3 h of reperfusion. However, this decline was only statistically significant in the control group compared with baseline values. None of the rabbits had aortic stenosis or developed aortic regurgitation for the duration of the study. Moreover, both the control and sildenafil groups demonstrated a comparable amount of mitral regurgitation (no more than mild) after ischemia/reperfusion, and none of the rabbits had baseline mitral regurgitation.

#### **2.5 Sildenafil and vardenafil preserve LV function in female mice**

Since the impact of PDE-5 inhibitors on the female cardiovascular system following ischemia remains unknown, we interrogated the effect of sildenafil and vardenafil on ischemia/reperfusion injury in female mice. In this study, adult female mice were pretreated (ip, bid) with sildenafil (0.7 mg/kg), vardenafil (0.14 mg/kg) or saline one hour before left coronary artery ligation for 30 minutes and reperfusion for 24 hours (Salloum et al., 2008b). Cardiac function, evaluated using echocardiography, showed that LV enddiastolic and end-systolic diameters increased 7 days post myocardial infarction with saline

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 105

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

(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 ischemia/reperfusion injury in women with coronary artery disease.
