Sildenafil Recovers Burn-Induced Cardiomyopathy

Abstract

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.

Keywords: PDE5A; burn injury; cardiomyopathy; fibrogenesis; oxidative stress; sildenafil.

Figure 1

The importance of the PDE5A-cGMP-PKG pathway in cardiomyocytes after a burn. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart tissues and blood were harvested at 24 h post burn (24 hpb ± SIL). (A) Representative LC MS/MS heat map; green color indicates down-regulated proteins, black color indicates unchanged proteins and red indicates up-regulated proteins (A,a). Corresponding expression levels of PKG (A,b), PDE5A (A,c.) and COL3A1 (A,d). (B) Myocardial levels of PDE5 mRNA by qRT-PCR (panel a), cyclic GMP (cGMP) protein levels in heart tissue (panel b) and PKG activity in heart tissue (panel d). Data are presented as mean value ± SD, shown as * (24 hpb vs. matched sham control) or ^& (24 hpb vs. 24 hpb/SIL), and presented as ***^{, &&&} p < 0.001 (n = ≥ 6 per group).

Figure 2

Effect of PDE5A inhibition on burn-induced cardiac dysfunction. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart function was measured using ECHO (Vevo^® 2100 System) at 24 h post burn (24 hpb ± SIL). Shown are (A) cardiac output (CO), (B) ejection fraction (EF), (C) stroke volume (SV), (D) left ventricular posterior wall at diastole (LVPW), (E), fractional shortening (FS) and (F) left ventricular systolic volume (LV Vol.). Data are presented as mean value ± SD, shown as ^{**, &&} p < 0.01, ***^{, &&&} p < 0.001 (n = ≥ 6 per group).

Figure 3

Effect of PDE5 inhibition on burn-induced cardiac fibrogenesis. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart tissues and blood were harvested at 24 h post burn (24 hpb ± SIL). (A) Representative myocardial fibrosis levels via Masson’s trichrome staining (panels a–d). (B) Representative whole hearts stained with Masson’s trichrome and scored for fibrosis (panel e). (C) Quantitative RT-PCR analysis of mRNA levels for fibrosis markers. Shown are atrial natriuretic peptide (panel a, ANP), natriuretic peptide B (panel b, BNP), collagen isoforms COLIA2 and COL IIIA1 (panels c & d), actin, alpha, cardiac muscle 1 (aSMA, panel e), and actin, alpha 2, smooth muscle, aorta (panel f). Results were normalized to rat GAPDH and β-actin mRNAs, and represent fold change after the burn (±SIL), as compared to that noted in matched normal controls. In all figures, data are plotted as mean value ± SEM (n ≥ 6 per group). Significance is shown as * (24 hpb vs. matched control) or ^& (24 hpb/untreated vs. 24 hpb/SIL), and presented as ***^,&&& p < 0.001 (n = ≥ 6 per group).

Figure 4

Effect of PDE5 inhibition on myocardial inflammation after a burn. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart tissues and blood were harvested at 24 h post burn (24 hpb ± SIL). (A) H&E staining of the left ventricle (magnification: 20×. Pink: muscle/cytoplasm/keratin, blue: nuclear) (panels a–d), (B) H&E staining of a whole heart (magnification: 20×. Purple: muscle/cytoplasm/keratin, blue: nuclear), and scored for inflammation (C). (D) Shown are the myocardial levels of inflammation cytokine mRNA levels determined by real time PCR, including RELA (a), IL-18 (b) and TGF-β (c). 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).

Figure 5

The effect of PDE5A inhibition on downstream gene expression after a burn. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart tissues and blood were harvested at 24 h post burn (24 hpb ± SIL). Isolated heart tissue RNAs (RNeasy Mini Kit) were used to synthesize cDNAs (The SuperScript III First-Strand Synthesis System), and mRNA level was measured by qRT-PCR. (A) Myocardial levels of PKG mRNA. (B) Myocardial levels of cGMP-PKG pathway markers, including IRAG (a) and PLB (b) mRNA levels. (C) Myocardial level of RGS2 mRNA. (D) Myocardial level of RhoA mRNA. (E) Myocardial levels of MYTP mRNA levels. 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).

Figure 6

The effect of PDE5 inhibition on oxidant/antioxidant imbalance. Sprague–Dawley rats were randomized to a 60% TBSA scald burn or sham procedure with standard resuscitation with/without sildenafil (SIL). Heart tissues and blood were harvested at 24 h post burn (24 hpb ± SIL). (A) Shown are H₂O₂ levels (a), MDA levels (b) and protein carbonylation (c) in myocardium after burn. (B) Shown are cardiac antioxidants, including total antioxidant (a), total SOD activity (b) and Cu,ZnSOD activity (c) after burn. Results were normalized to rat GAPDH and β-actin mRNAs, and represent fold change after 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 < 0.01, ***^{, &&&} p < 0.001 (n = ≥ 6 per group).