Phosphodiesterase 5a Inhibition with Adenoviral Short Hairpin RNA Benefits Infarcted Heart Partially through Activation of Akt Signaling Pathway and Reduction of Inflammatory Cytokines

Introduction Treatment with short hairpin RNA (shRNA) interference therapy targeting phosphodiesterase 5a after myocardial infarction (MI) has been shown to mitigate post-MI heart failure. We investigated the mechanisms that underpin the beneficial effects of PDE5a inhibition through shRNA on post-MI heart failure. Methods An adenoviral vector with an shRNA sequence inserted was adopted for the inhibition of phosphodiesterase 5a (Ad-shPDE5a) in vivo and in vitro. Myocardial infarction (MI) was induced in male C57BL/6J mice by left coronary artery ligation, and immediately after that, the Ad-shPDE5a was injected intramyocardially around the MI region and border areas. Results Four weeks post-MI, the Ad-shPDE5a-treated mice showed significant mitigation of the left ventricular (LV) dilatation and dysfunction compared to control mice. Infarction size and fibrosis were also significantly reduced in Ad-shPDE5a-treated mice. Additionally, Ad-shPDE5a treatment decreased the MI-induced inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β1, which was confirmed in vitro in Ad-shPDE5a transfected myofibroblasts cultured under oxygen glucose deprivation. Finally, Ad-shPDE5a treatment was found to activate the myocardial Akt signaling pathway in both in vivo and in vitro experiments. Conclusion These findings indicate that PDE5a inhibition by Ad-shPDE5a via the Akt signal pathway could be of significant value in the design of future therapeutics for post-MI heart failure.


Introduction
Chronic heart failure is the leading cause of mortality and morbidity worldwide, and is commonly a result of myocardial infarction (MI)-induced remodeling of the left ventricle (LV), which is characterized by LV dilatation and cardiac dysfunction [1,2]. There is therefore a critical need for therapies that effectively inhibit LV remodeling and preserve cardiac function in order to improve the clinical outcome of patients after MI.
The cyclic nucleotide cGMP plays a central role in cardiovascular regulation, influencing function, gene expression, and morphology [3]. Cardiac cGMP is hydrolyzed by members of phosphodiesterase (PDE) family [4] of which PDE5a acts more specifically [5]. PDE5a is a cytosolic protein and its inhibition was reported to benefit patients with pulmonary hypertension [6]. Myocardial PDE5a expression has been shown to increase in patients with advanced heart failure and contribute to LV remodeling subsequent to myocardial infarction [7]. Several previous studies have demonstrated the beneficial effects of PDE5a chemical inhibitors on heart disease, including the commercially offered drugs sildenafil, tadalafil and vardenafil [8][9][10]. Our recent study focused on prolonging the effective inhibition of PDE5a, and results demonstrated that PDE5a inhibition using an adenoviral vector inserted into the shRNA sequence could improve cardiac function and remodeling [11], as well as increase capillary density and capillary/myocyte ratio in our MI mouse model.
In addition, evidence from both animal and human studies suggests that increased inflammatory cytokines are associated with a poor prognosis following MI and may play an important role in the pathogenesis and progression of heart failure. Cytokines can influence heart contractility by inducing hypertrophy and promoting apoptosis or fibrosis, thereby contributing to the continuous myocardial remodeling process [12][13][14]. In pelvic ganglia neurons, for instance, a recent study has shown that PDE5 inhibition attenuates inflammation and oxidative stress after bilateral cavernosal nerve damage [15].
Our aims in the present study were to confirm the beneficial effects of PDE5a inhibition with Ad-shPDE5a on chronic post-MI heart failure, investigate the role of inflammatory cytokines involved in those effects, and characterize the molecular signaling upstream of the outcomes.

Animal Experimental Protocols
All animal work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). This study was approved by the IACUC at University of Cincinnati (Protocol Number: 06-03-03-01).
Humane endpoints during the animal survival study were identified as animals with weight loss of 20% or greater and/or animals with respiratory distress after opening of thoracic cavity surgery. If symptoms were to occur, veterinary staff was consulted and appropriate medical treatment was provided. If animals did not respond to appropriate treatment, animals were euthanized.
The animals were euthanized under anesthesia by cardioplegic solution injected into the heart to arrest the heart at diastolic phase followed by removal of vital organ.
Animals were monitored on a daily basis with assistance of the LAMS Veterinary Staff in evaluation the health of the animals. There were no unexpected deaths for the duration of the study.
Animal's pain/distress was monitored to ensure that the analgesic is effective by watching for guarding (protecting painful area), licking, biting, scratching, or shaking the painful area, restlessness, lack of normal interest in surroundings, failure to groom, abnormal postures, and lack of mobility.
MI was induced in 10-week-old male C57BL/6J mice by ligation of the left coronary artery as previously described [16]. Immediately after that, adenoviral Ad-shPDE5a (1x10 10 particles) was injected into multiple sites per mouse heart along the anterior and posterior left ventricular wall. As a control, adenoviral Null (Ad-Null) or DMEM culture medium (DMEM) was injected in the same manner. In sham-operated mice (Sham), the suture was passed but not tied.
MI mice were randomly assigned to the Ad-shPDE5 (n = 12), Ad-Null (n = 15) or DMEM (n = 12) treatment group, and followed up for 4 weeks. Sham-operated mice (n = 6) were injected with the same volume of DMEM in a similar manner and examined 4 weeks later.

Preparation and Transduction of Cultured Myofibroblasts
Myofibroblasts were isolated from 1-day-old neonatal C57BL/6J mice as reported previously [17]. Briefly, left ventricle tissue was excised, washed with HBSS and cut into pieces of 1~2 mm. Enzymatic digestion using Collagenase Type II (100 U) and 0.125% trypsin-EDTA was employed to dissociate the tissue pieces, which were agitated at 37°C for 40~60 min. Supernatant was collected every 10 min into a conical tube and digestion was neutralized with 1/10 volume of fetal bovine serum. Finally, the cells were centrifuged at 1,200 rpm for 5 min, resuspended in fresh medium (DMEM containing 10% FBS) and plated into 25 cm2 culture flasks. Non-adherent cells were discarded after 60 min. The remaining cells were propagated 4~5 passages, which then spontaneously induced into myofibroblasts. The myofibroblasts were plated on dishes and incubated in DMEM containing 10% FBS at 37°C for 24 h. The cells were exposed to infection medium containing 1x10 8 Ad-vector particles/ml for 16 h, followed by maintenance in normal medium for 56 h. The cells were used for biochemical analyses. Proteins extracted from myofibroblasts were used for Western blotting.
Myofibroblast culture medium was replaced with glucose-and serum-free DMEM for oxygen and glucose deprivation (OGD), and the cells were kept in an airtight anoxia chamber saturated with 95% N2/5% CO2. After 4 h or 8 h of incubation under oxygen and glucose deprivation, proteins were extracted from myofibroblasts and used for Western blot and ELISA.

Cardiac Function Studies
Animals were anesthetized via intraperitoneal injection with pentobarbital. Echocardiograms were then recorded 4 weeks post-MI, using an echocardiographic system (HDI-5000 SONOS CT) equipped with Compact Linear Array probe CL10-5. The heart was imaged in M-Mode, and recordings were obtained from parasternal long-axis view at the papillary muscle level.

Histological Analysis
After the physiological studies were completed, all surviving mice were euthanized and their hearts were removed. For measurement of infarction size and area of fibrosis, the heart was arrested in diastole by intravenous injection of cadmium chloride and fixed using formalin. The heart was then excised, cut transversely, and embedded in paraffin. Histological sections of 4-μm thickness were cut and used for hematoxylin-eosin and Masson's trichome staining for visualization of muscle architecture and thickness of the LV wall as described in the previous study [18]. Fibrosis and total LV area of each image were measured using the Image-Pro Plus (Media Cybernetics).

Western blot and ELISA
Proteins from heart tissues and cultured myofibroblasts were used for Western blot analysis. Proteins were separated and transferred to membranes by standard protocols, after which they were probed with antibodies against p-Stat3, p-Stat5, p-Akt, and p-Erk (all from Cell Signaling) and transforming growth factor (TGF-β1) (Promega). Three to five hearts from each group or cultured myofibroblasts were subjected to the Western blot. The blots were visualized by means of chemiluminescence (ECL, Thermo), and the signals were quantified by densitometry. α -Tubulin (analyzed with an antibody from Thermo) served as the loading control.
Levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the myocardium or cultured myofibroblasts were assayed with an ELISA (CycLex). Three to five hearts from each group or proteins extracted from myofibroblasts were used for this assay.

Statistical Analysis
Statistical analysis was performed using SPSS 17.0 software. Values are shown as mean ± SEM. The significance of differences between groups was evaluated with one-way ANOVA followed by the Newman-Keuls multiple comparison test. Values of P<0.05 were considered significant.

PDE5a Inhibition by Ad-shPDE5a in Cultured Myofibroblasts
Successful transduction of myofibroblasts with Ad-shPDE5a was performed for abrogation of PDE5a expression (Fig 1A). Western blot showed that 16 h transduction with Ad-shPDE5a significantly inhibited PDE5a expression in the myofibroblasts until days 3 or 6 of observation ( Fig 1B).

Effects of Ad-shPDE5a Treatment on Cardiac Function and Morphology at Chronic Stage of MI
Western blot analysis showed that Ad-shPDE5a treatment significantly decreased PDE5a levels in the infarcted hearts at both 1 wk and 4 wk after treatment compared with Ad-Null-treated animal hearts (Fig 3A). Four wk after coronary artery ligation, 5 (33%) of the Ad-Null, 3 (37%) of DMEM, and 1 (8%) of the AdshPDE5a-treated mice died. The survival rate 4 wk post-myocardial infarction (post-MI) was 67% in Ad-Null-treated and 92% in Ad-shPDE5a-treated animals (p = 0.11, Fig 3B).  Compared with 67% in the Ad-Null treated group, the survival rate was 92% in the Ad-shPDE5a-treated group (P = 0.11) (C) echocardiographic data for LV geometry and function 4 wk after treatment with Ad-Null and Ad-shPDE5a. LV diastolic diameter (LVDd), Echocardiography carried out 4 wk post-MI revealed that Ad-Null MI mice had marked enlargement of the LV cavity and reduced cardiac function when compared to the sham-operated mice, as indicated by increased LV end-systolic and end-diastolic diameter and reduced LV fractional shortening and ejection fraction (Fig 3C). All of these functional parameters were significantly attenuated in Ad-shPDE5a-treated mice (Fig 3C), suggesting that Ad-shPDE5a treatment mitigated post-MI remodeling and cardiac dysfunction through PDE5a inhibition. There was no significant difference in cardiac function 4 wk post-MI between the Null and DMEM-treated mice, indicating a negligible effect of Null treatment on myocardial function.
Ad-Null or DMEM-treated mice showed marked LV dilatation with a thin infarcted segment 4 wk post-MI, while Ad-shPDE5a-treated mice showed substantially smaller LV cavities and thicker infarcted segments (Fig 4A). Ad-Null and DMEM groups showed disintegrating myocytes within the center of the scar tissue, while abundant viable islands of myocytes were observed in the center of the infarct in the Ad-shPDE5a-treated group (Fig 4B). The size of the infarct was significantly reduced in Ad-shPDE5a-treated animals (25.4±2.9%, P<0.05) compared with Ad-Null (42.0±3.2%) and DMEM groups (43.3±2.6%). Severe fibrosis of the myocardium was observed in the Ad-Null (34.6±2.2) and DMEM groups (35.1±2.5), compared to that of the Ad-shPDE5a treated animals (26.5±1.9, P<0.05) (Fig 4C).

Discussion
Rapid recanalization of the occluded coronary artery is presently the best clinical approach for the treatment of acute MI, but not practical given the small window of therapeutic effectiveness in the few short hours after the onset of MI [19]. Patients who escape death during the acute stage of a large MI are also at high risk of developing heart failure during the later chronic stage [20]. Currently available therapies for post-MI heart failure are underdeveloped and underwhelming, creating great demand for an effective treatment.
PDE5a inhibition prevents the breakdown of cGMP into 5'-GMP that leads to cGMP accumulation. This attribute is currently being exploited as a novel strategy in cardiovascular therapeutics as a method of preventing cardiac remodeling and attenuation of cardiac fibrosis [21]. PKG is a serine/threonine protein kinase and is one of the major intracellular receptors for cGMP. It has been reported that the cGMP/PKG pathway has anti-apoptotic effects on cardiac cells [22]. Accumulation of cGMP in response to PDE5a inhibition counters the ill-effects of adrenergic stimulation and prevents cardiac hypertrophy and pressure-induced remodeling of the heart [23].
RNA interference as an innate biological phenomenon is achieved by loading the RNA interference silence complex with a short single-stranded antisense RNA that is complementary to a target mRNA [24]. For a longer-term inhibition of PDE5a in vivo, we constructed a PDE5a-specific shRNA adenoviral vector [11]. Compared with other commercial Phosphodiesterase 5a Inhibition in Infarcted Heart pharmacological PDE5a inhibitors (Sildenafil, Tadalafil etc.), PDE5a inhibition through Ad-shPDE5a offers the advantage of extended time duration of the protective effects and eliminates the need for repeated treatments.
In the present study, PDE5a enzyme activity was significantly inhibited in the LV after Ad-shPDE5a treatment. This resulted in a significant attenuation of fibrosis, which can contribute to both systolic and diastolic dysfunction [25] subsequent to ischemic episodes. Ad-shPDE5a treatment also mitigated LV remodeling, which was evident from the preserved LV dimensions during both systole and diastole. Finally, Ad-shPDE5a treatment significantly contributed to a reduction in infarction size and preserved myocardial function by maintenance of the viability of the residual cardiac myocytes.
Our previous study provided evidence that post-infarction interference therapy with short hairpin RNA targeting PDE5a relieved the adverse effects on LV geometry and function during the chronic stage [11] of MI. The present study builds on those findings demonstrating that Ad-shPDE5a increases cardiac function and reduces infarction size and fibrosis, revealing that Ad-shPDE5a reduces inflammatory cytokine production in the failing myocardium. Several studies have shown that in both animals and human beings with failing hearts, levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) are increased in plasma [13] as well as in the myocardium itself [26,14]. In the present study, levels of IL-1β, IL-6, TNF-α, and TGF-β were elevated in the failing myocardium of mice four weeks post-MI, consistent with those studies. Building upon that, our research revealed that Ad-shPDE5a treatment remarkably decreased the MI-induced inflammatory cytokines: interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transformed growth factor-β1. Expression of these inflammatory cytokines has been reported to directly relate to the degree of heart failure and relate inversely to survival [14,27]. Moreover, the results of animal studies and some clinical pilot trials have suggested that suppression of inflammatory cytokines may improve cardiac performance [28,29]. It is therefore conceivable that reduction of inflammatory cytokines may be one of the mechanisms involved in generating the beneficial effects of Ad-shPDE5a on IM-induced failing hearts.
Phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway can be activated by a variety of extracellular stimuli and the actions of Akt in the cell are numerous and diverse, but all result in anti-apoptosis or pro-cell proliferation effects [30]. cGMP is a downstream second messenger of nitric oxide (NO). In cardiac myocytes, the physiological effects of cGMP are exerted through the activation of protein kinase G (PKG) signaling. Previous studies have shown that NO inhibits apoptosis of retinal neurons in culture through the canonical cGMP/PKG-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3' kinase (PI3k) and Akt [30].Recent studies also showed that the activation of PI3K/Akt is involved in cell survival [31] and axonal outgrowth [32] in neurons. It has been reported that astrocytic Akt phosphorylation might be enhanced through eNOS/sGC/PKG/PI3K pathway, which increased activation of astrocytic Akt may result in the upregulation of pro-survival transcription factor and neuroprotective factor expression [33]. Activated eNOS produces of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). The above findings suggest that regulation of PI3K/Akt signaling pathway via activating cGMP/PKG-dependent pathway. This is consistent with our findings that phosphorylated Akt was significantly upregulated in the Ad-Null mice 4-weeks post-MI, and suggests that PDE5a inhibition by Ad-shPDE5a plays a key role in the underlying molecular mechanism for the pro-survival effects, given that AdshPDE5a treatment upregulates Akt signaling pathway via activating cGMP/PKG-dependent pathway. These physiological roles of Akt include involvement in metabolism, protein synthesis, apoptosis pathways, transcription factor regulation, and the cell cycle. Akt exerts its effects in cells by phosphorylating a variety of downstream substrates. Recent studies showed that the activation of PI3K/Akt is involved in cell survival [31] and axonal outgrowth [32] in neurons. These results suggest that NO from eNOS might regulate astrocytic Akt phosphorylation through NO/cGMP/PKG pathway [33]. This is consistent with our findings that phosphorylated Akt was significantly upregulated in the Ad-Null mice 4-weeks post-MI, and suggests that PDE5a inhibition by Ad-shPDE5a plays a key role in the underlying molecular mechanism for the pro-survival effects, given that Ad-shPDE5a treatment upregulates Akt signaling pathway.

Innovation
PDE5a inhibition with adenoviral short hairpin RNA produced beneficial effects on infarcted mouse hearts that included reduction of infarction size and fibrosis, and an overall improvement of cardiac function and remodeling. The underlying mechanism almost certainly is due in part to activation of the Akt signaling pathway and reduction of inflammatory cytokines, but more research is needed to define that relationship more clearly. These findings indicate that PDE5a inhibition by Ad-shPDE5a could be of significant importance in the design of future therapeutics for post-MI heart failure.