Sildenafil Recovers Burn-Induced Cardiomyopathy

Background: Severe burn injury initiates a feedback cycle of inflammation, fibrosis, oxidative stress and cardiac mitochondrial damage via the PDE5A-cGMP-PKG pathway. Aim: To test if the PDE5A-cGMP-PKG pathway may contribute to burn-induced heart dysfunction. Methods: Sprague–Dawley rats were divided four groups: sham; sham/sildenafil; 24 h post burn (60% total body surface area scald burn, harvested at 24 h post burn); and 24 h post burn/sildenafil. We monitored heart function and oxidative adducts, as well as cardiac inflammatory, cardiac fibrosis and cardiac remodeling responses in vivo. Results: Sildenafil inhibited the burn-induced PDE5A mRNA level and increased the cGMP level and PKG activity, leading to the normalization of PKG down-regulated genes (IRAG, PLB, RGS2, RhoA and MYTP), a decreased ROS level (H2O2), decreased oxidatively modified adducts (malonyldialdehyde [MDA], carbonyls), attenuated fibrogenesis as well as fibrosis gene expression (ANP, BNP, COL1A2, COL3A2, αSMA and αsk-Actin), and reduced inflammation and related gene expression (RELA, IL-18 and TGF-β) after the burn. Additionally, sildenafil treatment preserved left ventricular heart function (CO, EF, SV, LVvol at systolic, LVPW at diastolic and FS) and recovered the oxidant/antioxidant balance (total antioxidant, total SOD activity and Cu,ZnSOD activity). Conclusions: The PDE5A-cGMP-PKG pathway mediates burn-induced heart dysfunction. Sildenafil treatment recovers burn-induced cardiac dysfunction.


Introduction
Globally, burns are a serious public health problem. An estimated 265,000 deaths occur each year from fires alone, with more deaths from scalds, electrical burns and other forms of burns, for which global data are not available [1]. Every day, over 300 children aged 0 to 19 are treated in emergency rooms for burn-related injuries, and two children die as a result of being burned [2]. In the United States, burns are also a considerable health problem, with 500,000 injured people, resulting in more than 40,000 hospitalizations, 4000 deaths and more than $18 billion in costs each year [3,4]. Burn injury causes hemodynamic derangements and compromised heart function, leading to organ hypoperfusion, burn zone extension and increased susceptibility to wound infection [5].
In cardiomyocytes, atrial natriuretic peptide (ANP) and nitric oxide (NO) produce cyclic guanosine monophosphate (cGMP) by activating guanylyl cyclase (GC), which stimulates cGMP-dependent protein kinase (PKG1α) [6]. PKG1α maintains the contractile force of cardiomyocytes and phosphorylates serine and threonine residues on numerous cytosolic proteins [7]. The cGMP-PKG axis regulates the activation of phosphodiesterase, which hydrolyzes cyclic nucleotide monophosphate (cNMP) [8]. While phosphodiesterases (PDEs) have seven isoforms in myocytes (including PDE1, 2, 3, 4, 5, 8, and 9), PDE5A is cardiomyocyte-specific, and functions to hydrolyze cGMP and negatively chromatography system (UltiMate 3000 RSLCnano, Dionex, Sunnyvale, CA, USA) coupled online to a Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) through a nano-spray ion source (Thermo Scientific). The analytical column was an Acclaim PepMap 100 (75 µm × 25 cm) from Thermo Scientific. After equilibrating the column, the samples (1 µL in solvent A) were injected onto the trap column and subsequently eluted (400 nL/min) by gradient elution onto the C18 column. All LC-MS/MS data were acquired using XCalibur, version 2.1.0 (Thermo Fisher Scientific), in positive ion mode, using a top speed data-dependent acquisition (DDA) method with a 3 s cycle time. The survey scans (m/z 350-1500) were acquired in the Orbitrap at 120,000 resolution (at m/z = 400) in profile mode, with a maximum injection time of 50 ms and an AGC target of 400,000 ions. The S-lens RF level was set to 60. Isolation was performed in the quadrupole with a 1.6 Da isolation window, and CID MS/MS acquisition was performed in profile mode using a rapid scan rate with detection in the orbitrap (res: 35,000) with the following settings: parent threshold = 5000; collision energy = 35%; maximum injection time 100 ms; AGC target 500,000 ions. Monoisotopic precursor selection (MIPS) and charge state filtering were on, with charge states 2-6 included. Dynamic exclusion was used to remove selected precursor ions, with a ±10 ppm mass tolerance, for 60 s after acquisition of one MS/MS spectrum. Database Searching: Tandem mass spectra were extracted and charge state deconvoluted with Proteome Discoverer (Thermo Fisher, version 1.4.1.14). All MS/MS spectra were searched against a Uniprot Rattus database (version 05-16-2017) using Sequest. Searches were performed with a parent ion tolerance of 5 ppm and a fragment ion tolerance of 0.60 Da. Trypsin was specified as the enzyme, allowing for two missed cleavages. Fixed modification of carbamidomethyl (C) and variable modifications of oxidation (M) and glycosylation were specified in Sequest.

cGMP Level
Weighted heart tissues were homogenized on ice (5-10 mL of 5% trichloroacetic acid (TCA)/per gram of tissue). After centrifugation at 1500× g for 10 min, TCA was extracted five times from the supernatant with water saturated ether. The aqueous phase was dried under a stream of nitrogen and resuspended in 1.5 mL of phosphate buffer. cGMP levels were measured by ELISA (variability among triplicate values, 10%). The values of cGMP in blank were subtracted, and the results were expressed as pmol/mg for tissues.

The cGMP Dependent Protein Kinase (PKG) Activity
A CycLex ® cGK (PKG) ELISA Assay Kit (MBL International Corp, Woburn, MA, USA) was employed to measure the PKG activity. Briefly, tissue homogenates (10 µg protein/10 µL) were added to 96-well plates pre-coated with histone H1 peptide containing threonine residues, and sequentially incubated for 30 min in the presence of cGMP and ATP, and then for 60 min with HRP-conjugated anti-phospho-G-kinase substrate threonine 68/119 monoclonal antibody. The plates were then washed, and HRP catalyzed conversion of chromogenic TMB substrate to blue color was recorded at 450/540 nm (standard curve: 1-10-units recombinant cGK [PKG] protein).

Echocardiography (ECHO)
Rats were sedated with inhalant anesthesia (1.5% isoflurane/100% O 2 ) and placed supine on an electrical heating pad at 37 • C, and heart rate and respiratory physiology were continuously monitored by ECHO. After shaving the chest, warmed ultrasound gel was applied, and transthoracic ECHO was performed using the Vevo ® 2100 ultrasound system (VisualSonics, Toronto, On, Canada) equipped with a high-frequency linear array transducer (MS250 13-24 MHz) [17]. All measurements were obtained in triplicate, and data were analyzed using the Vevo ® 2100 standard measurement package.

Histology
Tissue sections were fixed in 10% buffered formalin for at least 24 h, dehydrated in absolute ethanol, cleared in xylene and embedded in paraffin. For whole heart tissue, after extracting blood Cells 2020, 9, 1393 4 of 16 from the heart, the heart was perfused through the aorta by giving 50 mL of 10% buffered formalin. Five-micron tissue sections were subjected to staining with hematoxylin and eosin (H&E) or Mason's Trichrome at the Research Histopathology Core at the UTMB. The images were obtained by Motic EasyScanner and analyzed using Motic DSAssistant software (Motic North America, Richmond, British, BC, Canada) and Image J software. Myocarditis (presence of inflammatory cells) in H&E stained tissue sections was scored as 0 (absent), 1 (focal/mild, ≤1 foci), 2 (moderate, ≥2 inflammatory foci), 3 (extensive coalescing of inflammatory foci or disseminated inflammation), or 4 (diffuse inflammation, tissue necrosis, interstitial edema and loss of integrity). Inflammatory infiltrates were characterized as 'diffuse' or 'focal', depending upon how closely the inflammatory cells were associated [18]. Fibrosis was assessed by measuring the collagen area as a percentage of the total myocardial area, and categorized as (0) <1%, (1) 1-5%, (2) 5-10%, (3) 10-15%, and >15%, based on percentage of fibrotic area [19].

Gene Expression Analysis
Heart tissue-sections (10 mg) were homogenized in 100 µL TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted and precipitated by the chloroform/isoamyl alcohol/isopropanol method. The RNAs were treated by DNase I, RNase-free (Westlake, LA, USA. Cat# M0303S) to digest contaminated genomic DNA. To judge the integrity and overall quality of isolated RNAs, 2 µg RNAs were added to 10× native agarose gel loading buffer (15% ficoll, 0.25% xylene cyanol, 0.25% bromophenol blue) and run on 1% native agarose gels. The integrity RNAs were washed with 75% cold ethanol, re-suspended in 20 µL of UltraPure™ nucleotide-free distilled water, and a DU ® 700 UV/Visible Spectrophotometer (Beckman Coulter, Pasadena, CA, USA) was used to measure absorbance at 260 and 280 nm (OD 260/280 ratio ≥ 2, 1 OD 260 Unit = 40 µg/mL RNA). Total RNA (2 µg) was reverse transcribed with Oligo(dT) 20 primer using a SuperScript ® III Reverse Transcriptase (Invitrogen). The cDNA was used as a template in a real-time quantitative PCR on an iCycler Thermal Cycler with SYBR-Green Supermix (Bio-Rad, Hercules, CA, USA) and gene-specific oligonucleotides ( Table 1). The threshold cycle (Ct) values for target mRNAs were normalized to GAPDH/β-actin/α-tubulin mRNAs, and the relative expression level of each target gene was calculated as fold change [19].

Oxidative Stress
The protein carbonyls in the tissue homogenates were measured by colorimetric protein carbonyl assay (Cayman Chemicals, cat# 10005020), according to the instructions provided by the manufacturer. Protein carbonyl content is expressed in nmol/mg of protein. ROS release was measured by Amplex red assay. Briefly, homogenized samples of heart tissue (50 µg protein) were added to black flat-bottom plates. The reaction was initiated by the addition of 50 µL each of 100 µM Amplex red (Invitrogen) and Cells 2020, 9, 1393 5 of 16 0.3 U/mL HRP. The HRP catalyzed Amplex red oxidation by H 2 O 2 , resulting in fluorescent resorufin formation, was monitored at Ex 563 nm /Em 587 nm (standard curve: 50 nM-5 M H 2 O 2 ) using a Spectra Max M2 microplate reader (Molecular Devices, San Jose, CA, USA). A thiobarbituric acid-reactive substances (TBARS) assay was used to measure MDA-TBA adduct formation using the Cayman TBARS assay kit (cat# 700870). The MDA-TBA adduct was measured colorimetrically at 530-540 nm using a microplate reader (BioTek, Winooski, VT, USA). The MDA concentration is expressed in nM/mg protein.

ATP Colorimetric Assay
Tissues were homogenized on dry ice using the ice-cold ATP assay buffer provided as part of the ATP colorimetric Assay kit (Abcam, Cambridge, UK, cat# ab83355;). A 5 µL aliquot of the homogenized tissue was used to determine protein concentrations using the Thermo Scientific BCA method. ATP concentrations in the samples were calculated by plotting the measured optical densitometry at 570 nm in a microplate reader versus the linear distribution generated by the standard curve, with a final adjustment for protein concentration.

Statistical Analysis
All experiments were conducted with triplicate observations per sample (n = 6-9 rats/group) and data were expressed as mean ± standard error mean (SEM). All data were analyzed using GraphPad Prism8.30 software. The data (linear range or log10 transformed) were analyzed by the Kolmogorov-Smirnov test under column statistics to determine if the data are normally distributed. Normally distributed data were analyzed by Student's t-test (comparison of two groups) and one-way ANOVA with Tukey's test (comparison of multiple groups). If data were not normally distributed, then Mann-Whitney (comparison of two groups) and Kruskal-Wallis (K-W, comparison of multiple groups) tests were employed. Significance is presented by * 24 hpb vs. sham normal or & 24 hpb vs. 24 hpb/sildenafil (* ,& p < 0.05, ** ,&& p < 0.01, *** ,&&& p < 0.001).

The Importance of the PDE5A-cGMP-PKG Pathway in Cardiomyocytes after a Burn
To know what pathways would be involved in heart dysfunction after a burn, we first employed the Nano LC MS/MS approach to run proteomics (Figure 1(Aa)). Compared to the sham group, the burn group demonstrated a 32% decrease in PKG protein level (Figure 1(Ab)), a three-fold increase in PDE5A protein level (Figure 1(Ac)) and a ten-fold increase in COL3A protein level (Figure 1(Ad)). qPCR found a two-fold increase in PDE5A mRNA level (Figure 1(Ba)). Additionally, we found a two-fold decrease in cardiac cGMP levels (Figure 1(Bb)). Functionally, our data demonstrated a 32% decrease in PKG activity (Figure 1(Bc)). For confirmation of PDE5A-cGMP-PKG pathway involvement, sildenafil was administered after the burn. Sildenafil treatment normalized the levels of myocardial expression of PDE5A, cGMP level and PKG activity (Figure 1(Ba-c)). Altogether, these data suggest that burn-induced heart dysfunction occurs via the PDE5A-cGMP-PKG pathway.
the Nano LC MS/MS approach to run proteomics (Figure 1(Aa)). Compared to the sham group, the burn group demonstrated a 32% decrease in PKG protein level (Figure 1(Ab)), a three-fold increase in PDE5A protein level (Figure 1(Ac)) and a ten-fold increase in COL3A protein level (Figure 1(Ad)). qPCR found a two-fold increase in PDE5A mRNA level (Figure 1(Ba)). Additionally, we found a twofold decrease in cardiac cGMP levels (Figure 1(Bb)). Functionally, our data demonstrated a 32% decrease in PKG activity (Figure 1(Bc)). For confirmation of PDE5A-cGMP-PKG pathway involvement, sildenafil was administered after the burn. Sildenafil treatment normalized the levels of myocardial expression of PDE5A, cGMP level and PKG activity (Figures 1(Ba-c)). Altogether, these data suggest that burn-induced heart dysfunction occurs via the PDE5A-cGMP-PKG pathway.

Effect of PDE5 Inhibition on Burn-Induced Cardiac Fibrogenesis
Histologic examination of LV heart muscle pieces indicated a seven-fold increase in collagen accumulation (Figure 3(Aa,c); score: 4.023 ± 0.395 vs. 0.5 ± 0.04). To confirm that these results were not location specific, we fixed a whole heart, cut vertically to show all four chambers, and then performed Trichrome staining ( Figure 3B). Whole heart staining for fibrosis demonstrated that the burn injury resulted in cardiac fibrogenesis, as shown in Figure 3. Additionally, qPCR demonstrated approximately four-fold increase in ANP and BNP expression (Figure 3(Ca,b)), an approximately nine-fold increase in collagens I and III expression (Figure 3(Cc,d)), and a greater than three-fold increase in alpha cardiac smooth muscle actin1 (αSMA) and alpha cardiac smooth muscle actin 2 (ACTA) (Figure 3(Ce,f)) after the burn. Sildenafil treatment alleviated more than 90% of collagen deposits after the burn ( Figure 3A,B). Sildenafil treatment also normalized the mRNA expression levels of ANP and BNP, collagens I and III, αSMA and ACTA ( Figure 3C). These data suggest that treatment with sildenafil maintains the PDE5A-cGMP-PKG balance and has a cardio-protective role via inhibition of burn-induced interruption of the PDE5A-cGMP-PKG pathway. increase in alpha cardiac smooth muscle actin1 (αSMA) and alpha cardiac smooth muscle actin 2 (ACTA) (Figures 3(Ce,f)) after the burn. Sildenafil treatment alleviated more than 90% of collagen deposits after the burn (Figures 3A,B). Sildenafil treatment also normalized the mRNA expression levels of ANP and BNP, collagens I and III, αSMA and ACTA ( Figure 3C). These data suggest that treatment with sildenafil maintains the PDE5A-cGMP-PKG balance and has a cardio-protective role via inhibition of burn-induced interruption of the PDE5A-cGMP-PKG pathway.

Effect of PDE5A Inhibition on Myocardial Inflammation after a Burn
The cardiac inflammatory response plays an important role in the pathogenesis of cardiac tissue damage [20]. PDE5A inhibitors may increase cGMP levels, thus abrogating cardiac inflammation and cellular damage. Histologic studies exhibited diffuse inflammatory infiltrates in the myocardial tissue after the burn (H&E score: 2.812 ± 0.393 vs. 0.35 ± 0.039; Figures 4(Aa, c and C)). This was associated with a greater than five-fold increase in NF-κB and cytokines (RELA, 7.46-fold increase; IL-18, 5.03fold increase and TGF-β, 10.04-fold increase; Figures 4(Da-c)). Similar to Trichrome staining, H&E

Effect of PDE5A Inhibition on Myocardial Inflammation after a Burn
The cardiac inflammatory response plays an important role in the pathogenesis of cardiac tissue damage [20]. PDE5A inhibitors may increase cGMP levels, thus abrogating cardiac inflammation and cellular damage. Histologic studies exhibited diffuse inflammatory infiltrates in the myocardial tissue after the burn (H&E score: 2.812 ± 0.393 vs. 0.35 ± 0.039; Figure 4(Aa, c and C)). This was associated with a greater than five-fold increase in NF-κB and cytokines (RELA, 7.46-fold increase; IL-18, 5.03-fold increase and TGF-β, 10.04-fold increase; Figure 4(Da-c)). Similar to Trichrome staining, H&E staining of a whole heart provided the same results (Figure 4(Ba,c)). Sildenafil significantly reduced myocardial tissue inflammatory infiltrates (histological score: 0.905 ± 0.148; Figure 4(Ad, Bd and C)). NF-κB and cytokine expression was also normalized after sildenafil treatment ( Figure 4D). These data suggest that PDE5A inhibition was beneficial in regulating cardiac inflammatory infiltrates and cardiac tissue damage caused by severe burns.
staining of a whole heart provided the same results (Figures 4(Ba,c)). Sildenafil significantly reduced myocardial tissue inflammatory infiltrates (histological score: 0.905 ± 0.148; Figures 4(Ad, Bd and C)). NF-κB and cytokine expression was also normalized after sildenafil treatment ( Figure 4D). These data suggest that PDE5A inhibition was beneficial in regulating cardiac inflammatory infiltrates and cardiac tissue damage caused by severe burns. Results were normalized to rat GAPDH and β-actin mRNAs, and represent fold change after a burn (± SIL), as compared to that noted in matched normal controls. In all figures, data are plotted as mean value ± SD. Significance is shown as * (24 hpb vs. control) or & (24 hpb vs. 24 hpb/SIL), and data are presented as *** , &&& p < 0.001 (n = ≥ 6 per group).

The Effect of PDE5A Inhibition on Downstream Gene Expression after a Burn
To examine the role of PDE5A-regulated gene expression, we measured PDE5A-regulated gene mRNA levels. We found burn induced decreases of PKG (65%, Figure 5A), IRAG (90% Figure 5(B,a)), Results were normalized to rat GAPDH and β-actin mRNAs, and represent fold change after a burn (± SIL), as compared to that noted in matched normal controls. In all figures, data are plotted as mean value ± SD. Significance is shown as * (24 hpb vs. control) or & (24 hpb vs. 24 hpb/SIL), and data are presented as *** , &&& p < 0.001 (n = ≥ 6 per group).

The Effect of PDE5A Inhibition on Oxidant/Antioxidant Imbalance
Our recent publication [13] revealed that burn injury resulted in cardiac mitochondrial damage and transcription factor regulation of antioxidant gene expression, leading to oxidative stress. In this study, we measured oxidative stress markers to determine the role of PDE5 inhibition on ROS generation and the oxidant/antioxidant balance. We found a 16-fold increase in H2O2 (Figure 6(Aa)), a 3.4-fold increase in malondialdehyde (MDA; Figure 6(Ab)), and a 19-fold increase in the protein carbonyl level (Figure 6(Ac)) after a burn. Sildenafil treatment partially recovered H2O2 (50.59%) and MDA levels (73.33%), and completely normalized protein carbonyl levels ( Figure 6A). Total antioxidants (Figure 6(Ba)), total SOD activity ( Figure 6(Bb)), and Cu,ZnSOD (Figure 6(Bc)) showed declines of 60%, 87%, and 22% after a burn, respectively. Sildenafil administration normalized antioxidants, including total antioxidants (Figure 6(Ba)), total SOD activity ( Figure 6(Bb)) and Cu,ZnSOD activity ( Figure 6(Bc)). These observations suggest that sildenafil not only inhibited Results were normalized to rat GAPDH and β-actin mRNAs, and represent fold change after a burn (±SIL), as compared to that noted in matched normal controls. In all figures, data are plotted as mean value ± SD. Significance is shown as * (24 hpb vs. control) or & (24 hpb vs. 24 hpb/SIL), and presented as *** , &&& p < 0.001 (n = ≥ 6 per group).

The Effect of PDE5A Inhibition on Oxidant/Antioxidant Imbalance
Our recent publication [13] revealed that burn injury resulted in cardiac mitochondrial damage and transcription factor regulation of antioxidant gene expression, leading to oxidative stress. In this study, we measured oxidative stress markers to determine the role of PDE5 inhibition on ROS generation and the oxidant/antioxidant balance. We found a 16-fold increase in H 2 O 2 ( Figure 6(Aa)), a 3.4-fold increase in malondialdehyde (MDA; Figure 6(Ab)), and a 19-fold increase in the protein carbonyl level (Figure 6(Ac)) after a burn. Sildenafil treatment partially recovered H 2 O 2 (50.59%) and MDA levels (73.33%), and completely normalized protein carbonyl levels ( Figure 6A). Total antioxidants (Figure 6(Ba)), total SOD activity ( Figure 6(Bb)), and Cu,ZnSOD (Figure 6(Bc)) showed declines of 60%, 87%, and 22% after a burn, respectively. Sildenafil administration normalized antioxidants, including total antioxidants (Figure 6(Ba)), total SOD activity ( Figure 6(Bb)) and Cu,ZnSOD activity ( Figure 6(Bc)). These observations suggest that sildenafil not only inhibited PDE5A, but was also involved in the positive regulation of either total or mitochondria specific antioxidants. PDE5A, but was also involved in the positive regulation of either total or mitochondria specific antioxidants.

Discussion
In this study, we hypothesized that the NO-PED5-cGMP-PKG pathway plays a very important role in cardiac dysfunction after a burn injury, and that sildenafil, a PDE5A inhibitor, would ameliorate burn-induced cardiac dysfunction. Previous research demonstrated that the NO-PDE5A-cGMP-PKG pathway can preserve heart function and cardiomyocyte mitochondrial function through PKG1α kinase activation [21], and that PDE5A is upregulated in the hypertrophied heart [22,23]. Our data demonstrate that sildenafil protects against heart fibrogenesis and failure after a burn, prevents PKG1α kinase activity deficiency, increases ROS scavenging capacity, preserves oxidative protein adducts and abrogates cardiomyocyte inflammatory infiltrate after a burn. To the best of our knowledge, this is the seminal study demonstrating that the PDE5A-cGMP-PKG pathway plays a central role in burn-induced heart dysfunction. This study is also the first to demonstrate that sildenafil preserves antioxidant scavenging capacity while arresting the oxidative and inflammatory infiltration that causes cardiomyocyte death and cardiac remodeling after a burn. Additionally, we propose that currently available PDE5A targeting drugs, such as sildenafil, might be useful in the treatment of patients with burn-induced cardiac dysfunction.
Previously published articles have shown that sildenafil is effective in numerous heart-related diseases, such as diabetes [24], Chagas disease [25], heart failure [26], hypertension [27] and burns [14]. Our previous work has demonstrated that a burn induces cardiac mitochondrial damage,

Discussion
In this study, we hypothesized that the NO-PED5-cGMP-PKG pathway plays a very important role in cardiac dysfunction after a burn injury, and that sildenafil, a PDE5A inhibitor, would ameliorate burn-induced cardiac dysfunction. Previous research demonstrated that the NO-PDE5A-cGMP-PKG pathway can preserve heart function and cardiomyocyte mitochondrial function through PKG1α kinase activation [21], and that PDE5A is upregulated in the hypertrophied heart [22,23]. Our data demonstrate that sildenafil protects against heart fibrogenesis and failure after a burn, prevents PKG1α kinase activity deficiency, increases ROS scavenging capacity, preserves oxidative protein adducts and abrogates cardiomyocyte inflammatory infiltrate after a burn. To the best of our knowledge, this is the seminal study demonstrating that the PDE5A-cGMP-PKG pathway plays a central role in burn-induced heart dysfunction. This study is also the first to demonstrate that sildenafil preserves antioxidant scavenging capacity while arresting the oxidative and inflammatory infiltration that causes cardiomyocyte death and cardiac remodeling after a burn. Additionally, we propose that currently available PDE5A targeting drugs, such as sildenafil, might be useful in the treatment of patients with burn-induced cardiac dysfunction.
Previously published articles have shown that sildenafil is effective in numerous heart-related diseases, such as diabetes [24], Chagas disease [25], heart failure [26], hypertension [27] and burns [14]. Our previous work has demonstrated that a burn induces cardiac mitochondrial damage, as evidenced by morphological changes on electron microscopy, cardiac mitochondrial replication deficiency, and decreased mitochondrial complex activity and oxygen consumption [14]. Interestingly, treatment with sildenafil preserved the mitochondrial structure, respiratory chain efficiency and energy status after a burn [14]. This previous work indicates that PDE5A inhibition could be beneficial in treating burn-induced heart dysfunction. However, the complete mechanism remained unclear, as did the question of clinical improvement. This is the first study to demonstrate that the use of a PDE5A inhibitor leads to functional improvement in burn-induced cardiac dysfunction, and that cardiac fibrosis, inflammation and oxidative stress can be mitigated as well.
Heart damage is a well-documented complication that enhances mortality and morbidity after a severe burn injury [28]. However, little is known about the role of the PED5A-cGMP-PKG pathway in burn-induced cardiac dysfunction. PDE5A activation depresses cGMP and PKG levels, whereas PDE5A inhibition would do the reverse [29]. Furthermore, cGMP converts inactive PKG to activated PKG [9], resulting in phosphorylation of the cell membrane and a decrease in cell membrane-bound protein Ras homolog member A (RhoA) activation. This adjusts Rho kinase (ROCK), RGS2 and myosin phosphatase targeting subunit (MYPT) [30]. PDE5A inhibition raises cGMP levels, leading to downstream effects on the heart and vasculature [31]. PDE5A inhibitors may also inhibit RhoA-Rho kinase [14]. In this study, sildenafil was seen to not only inhibit PDE5A, but also to affect PKG-regulated genes. Given this finding, future studies will examine whether sustained PKG activity would protect against burn-induced heart dysfunction.
Severe pediatric burn injury has a long-term effect on heart function into late adolescence and beyond, and is associated with myocardial fibrosis [32]. To test whether burn injury-induced heart function correlates with cardiac fibrosis, we measured heart fibrogenesis by staining heart tissues with trichrome. Our findings indicate that burn-induced heart dysfunction is associated with heart fibrogenesis, and that this process begins immediately after the initial burn injury. Interestingly, PDE5A inhibitor treatment ameliorated burn-induced myocardial fibrogenesis. Histologic and molecular studies demonstrated that burn injury results in cardiac fibrosis, widespread inflammatory infiltration and oxidative adducts in the heart tissue, all of which are common hallmarks of heart dysfunction. While the cause of cardiac dysfunction after a burn remains elusive, our results demonstrate that treatment with sildenafil has cardioprotective effects via preservation of systolic function after burn. We observed decreased heart wall thickness and fibrosis with sildenafil treatment after a burn, which may indicate a lasting protection against burn-induced cardiac dysfunction.
The canonical NF-κB pathway is activated by proinflammatory cytokines, resulting in the activation of RelA-containing complexes, which has an important role in the pathogenesis of chronic inflammatory diseases [33]. In burn-induced cardiac dysfunction, there are no previous studies that demonstrate the presence of RelA-containing complexes. Our data are the first to show that RelA expression in heart tissue is required for the recruitment of NF-κB after a burn (Figure 4(Da)), demonstrating that the canonical pathway of NF-κB plays a very important role in burn-induced inflammation. IL-18 primarily facilitates Th1-type immunoreactions, and helps to generate an inflammatory response [34]. Because IL-18 regulates the synthesis of TNF-α, IL-1β, IL-8 and MIP-1α, removal of IL-18 may have a beneficial effect in lethal endotoxemia in naive mice [35][36][37][38]. Previous studies also suggest that either mRNA or protein levels of IL-18 are enhanced in heart disease and heart infarction, and that IL-18 or IL-1β play a significant role in myocardial injury.
In ischemic cardiac disease, TGF-β neutralizes macrophages through Smad3-dependent pathways [39]. TGF-β may act as one of the key molecules in hypertrophy and heart failure, but its role in burn injury has yet to be elucidated [40]. Our study shows that burn injury increased TGF-β gene expression, and that the TGF-β system may be a promising therapeutic target for burn-induced cardiac dysfunction in the future.
PKG has pleiotropic physiological functions in the cardiovascular system [41]. Specifically, in terms of cardiomyocyte contractility, the substrates of PKG include myosin phosphatase subunit 1 (MYPT1), G-protein signaling 2 (RGS2), phospholamban (PLB) and inositol trisphosphate receptor-associated cGMP kinase substrate protein (IRAG), which demonstrates a functional regulation by PKG. To test the effects of a burn on the PKG pathway, we applied qPCR to measure related mRNA levels, and found significant decreases in PKG, IRAG, PLB, RGS2 and MYTP, as well as an increase in RhoA ( Figure 5). This indicates that burn-induced heart dysfunction is mediated via the PKG pathway, and that PDE5 inhibition is beneficial to preserve cardiac function after a burn injury.
ROS are correlated with enhanced PDE5A mRNA levels in cardiomyocytes during heart damage [42], but the mechanism remains unknown. Similarly, little is known about the influence of PDE5A inhibitors on oxidative damage and its association with the activation of cGMP-PKG. However, enhancement of cGMP may reduce NADPH oxidase expression or activity and, therefore, ROS production [43]. Our study shows that PDE5A is a key factor of the compromised antioxidant response, and that sildenafil treatment accelerates ROS scavenging capacity via activating SOD and enhancing total antioxidants. These findings uncover the fundamental cause for the imbalance between oxidants and antioxidants after a burn. The potentially therapeutic benefits of PDE5A inhibitors seem to be the host's capacity to avoid risks related to extreme levels of O 2 − that result in the overproduction of free radicals. Future studies will aim to illuminate whether PDE5A inhibits antioxidant capacity by targeting the PGC-1-α/NFE2L2 (Nuclear Factor, Erythroid 2 Like 2) pathway of antioxidant response in the heart. There is also significant overlap between the adrenergic and PDE5 pathways, which will require further study. Borlaug et al. demonstrated that sildenafil administration blunts systolic responses to β-adrenergic signaling, and that sildenafil inhibits β-adrenergic-stimulated cardiac contractility in humans [44]. Additionally, other work has demonstrated that PDE5 inhibition restores catecholamine responsiveness and partially reverses contractile dysfunction [45]. A burn stimulates much higher levels adrenergic proteins [46] and catecholamines [47] in the circulation. Given the interaction between these two systems, further study is needed to further define the interactions between these two pathways in burn-induced cardiac dysfunction.

Conclusions
Burn-induced cardiac dysfunction occurs via the PDE5A-cGMP-PKG pathway. The PDE5A inhibitor sildenafil has a potent cardio-protective effect, and acts by inhibiting cardiomyocyte inflammation, fibrogenesis and oxidative stress after a burn.