MG53 Protects against Sepsis-Induced Myocardial Dysfunction by Upregulating Peroxisome Proliferator-Activated Receptor-α

Figures

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  Figure 1. Protocol for experiments. CLP = cecal ligation and puncture; rhMG53 = recombinant human MG53.

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  Figure 2. Myocardial MG53 and PPARα decreased in SIMD rats. Supplementation of MG53 improved PPARα expression. (a) Representative western blot bands of MG53 and PPARα at 3 h, 6 h, 12 h, 18 h, and 24 h after CLP in experiment 1. Densitometric analysis of the (b) MG53 and (c) PPARα protein levels in experiment 1. (d) Representative western blot bands of MG53 and PPARα at 18 h after CLP in experiment 2. Densitometric analysis of the (e) MG53 and (f) PPARα protein levels in experiment 2. Data are presented as mean ± SD, n = 8. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human MG53.

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  Figure 3. MG53 improved cardiac function and survival rate in SIMD rats, which was exacerbated by GW6471. Cardiac function indexes were demonstrated by (a) heart rate (HR), (b) mean arterial blood pressure (MABP), (c) left ventricular systolic pressure (LVSP), (d) left ventricular end-diastolic pressure (LVEDP), and (e) maximum rate of rise/fall of left ventricle pressure (±dp/dtmax) in four groups. (f) Survival analysis. Data are presented as mean ± SD, n = 8 in cardiac function evaluation, n = 15 in survival rate evaluation. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human mitsugumin-53.

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  Figure 4. MG53 prevented cardiomyocyte injury in SIMD rats, which was reversed by GW6471. Myocardium architecture was visualized by (a) H&E staining (200x magnification, scale bar = 100 μm). Myocardial injury was detected through serum levels of (b) cardiac troponin I (cTnI), (c) creatine kinase isoenzyme MB (CK-MB), (d) lactate dehydrogenase (LDH), and (e) heart-type fatty acid-binding protein (H-FABP) by the ELISA method. Data are presented as mean ± SD, n = 8. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human mitsugumin-53.

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  Figure 5. MG53 attenuated myocardial oxidative stress in SIMD rats, which was abolished by GW6471. Oxidative stress injury was detected through (a) representative DHE stained images depicting in situ superoxide production (red, 400x magnification, scale bar = 50 μm), (b) quantification of DHE-signal intensity (folds of the sham group), (c) superoxide dismutase (SOD), (d) malondialdehyde (MDA), (e) glutathione (GSH) activity, and (f) free 15-F2t-isoprostane levels in the myocardium via an ELISA. Data are presented as mean ± SD, n = 8. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human mitsugumin-53.

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  Figure 6. MG53 alleviated myocardial inflammation in SIMD rats, which was exacerbated by GW6471. Collagen accumulation was demonstrated by (a) Masson's trichrome staining. Collagen was stained blue in paraffin-embedded heart tissue sections (400x magnification, scale bar = 50 μm) and (b) the quantitation of interstitial fibrosis area (%). Inflammatory cytokines (c) interleukin-1β (IL-β), (d) IL-6, and (e) tumor necrosis factor-α (TNF-α) in serum were detected by ELISA, and (f) cyclooxygenase-2 (COX-2) expression in the myocardium was detected by western blotting. Data are presented as mean ± SD, n = 8. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human mitsugumin-53.

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  Figure 7. MG53 prevents cardiomyocyte apoptosis in SIMD rats, which was abolished by GW6471. Cardiomyocyte apoptosis was assessed by (a) terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (400x magnification, scale bar = 50 μm) and (b) the quantitation of TUNEL-positive cells. TUNEL-positive cells were stained brown and are indicated by black arrows. (c) Representative western blot bands of pro-caspase-3, cleaved-caspase-3 (p17 form), Bax, and Bcl-2. Quantitative analysis of (d) the ratio of cleaved-caspase-3 (p17 form) to pro-caspase-3 and (e) the ratio of Bax to Bcl-2 in heart tissues. Data are presented as mean ± SD, n = 12. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CLP = cecal ligation and puncture; rhMG53 = recombinant human mitsugumin-53.

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  Figure 8. Schematic of the proposed mechanism involved in the protective effects of MG53 on PPARα in SIMD rats. Sepsis caused the H-FABP released from cardiomyocyte plasma and downregulated MG53 and PPARα protein levels. Endogenously, ROS were overproduced resulting in the activation of the caspase-3-dependent apoptosis pathway. Also, large amounts of inflammatory cytokines (IL-1, IL-6, and TNF-α) were generated due to the activation of some proinflammatory pathways (such as NF-κB) after SIMD. The supplementation of MG53 upregulated PPARα, attenuated cardiomyocyte damage, and prevented H-FABP release. H-FABP facilitated FAs transfer and activated PPARα. PPARα exhibited an anti-inflammatory effect through the inhibition of proinflammatory signaling pathways, such as NF-κB. Also, PPARα heterodimerized with RXR, banded to PPRE, and then recruited RNA Pol II to regulate gene function to play a crucial part in antioxidant stress, antiapoptosis, and promoting FAs metabolism. RXR = retinoid X receptor; RNA Pol II = RNA polymerase II; PPRE = PPAR response element; FAs = fatty acids.