Figure 1.
Time line illustrating the experimental design.
Figure 2.
Electron microscopic analysis of the LPS-treated rat heart.
Transmission electron micrographs of the rat heart treated with LPS (15 mg/kg, 3 h) with or without CoPPIX pretreatment (5 mg/kg, 24 h). Representative images in each group are shown: control (a), CoPPIX (b), LPS (c), LPS+CoPPIX (d). Bars, 500 nm. Black arrowheads indicate mitochondria swollen and/or surrounded by membranous structure. White arrowhead indicates mitochondria degraded within membranous structure.
Figure 3.
Effects of CoPPIX on autophagy and mitochondrial biogenesis during LPS treatment in the heart.
A, Western blot analysis of the rat heart treated with LPS (15 mg/kg for 1, 3, and 6 h) with or without CoPPIX pretreatment (5 mg/kg, 24 h) to determine the levels of LC3-II, p62, NRF1, TFAM, and HO1. Actin was detected as a loading control. B, Ratios between LC3, p62, NRF1, TFAM, HO1, and actin determined using densitometry analysis. Each bar represents the mean ± S.E. of four animals (**p<0.01; *p<0.05). Times indicated at the leftmost in the panel show the treatment times for LPS.
Figure 4.
Effects of SnPPIX on autophagy during LPS treatment in the heart.
Western blot analysis of the rat heart treated with LPS (15 mg/kg for 1, 3, and 6 h) with or without SnPPIX pretreatment (50 mg/kg, 24 h) to determine the levels of LC3-II and p62. Actin was detected as a loading control. Times indicated on the top of the panel show the treatment times for LPS.
Figure 5.
Effects of CoPPIX on lysosome reformation during LPS treatment in the rat heart.
A, Western blot analysis of the rat heart treated with LPS (15 mg/kg for 1, 3, and 6 h) with or without CoPPIX pretreatment (5 mg/kg, 24 h) to determine the levels of TFEB, LAMP1, and LAMP2. GAPDH was detected as a loading control. B, Ratios between TFEB, LAMP1, LAMP2, and GAPDH determined using densitometry analysis. Each bar represents the mean ± S.E. of four animals (**p<0.01). Times indicated at the leftmost side in the panel show the treatment times for LPS.
Figure 6.
Effects of CoPPIX on nuclear translocation and activation of TFEB during LPS treatment in the rat heart.
A, Immunohistochemical analysis of TFEB in the left ventricle of the rat heart at 3 h after LPS administration (15 mg/kg). Representative images in each group are shown: control (a), CoPPIX (b), LPS (c), LPS+CoPPIX (d). Bars, 5 µm. B, RT-PCR analysis of the LAMP1 and LAMP2 levels in the heart of LPS-treated and control animals for 1, 3, and 6 h. Amplification of actin cDNA was performed and served as the internal control. Each bar represents the mean ± S.E. of three animals (*p<0.05 vs. none).
Figure 7.
Histological and serological analyses of heart injuries during LPS administration in rats.
A and B, Formalin-fixed and paraffin-embedded sections (3-µm thickness) of rat heart tissues stained with H&E and with anti-4-HNE antibody. Representative H&E stains (A, ×400) and 4-HNE stains (B, ×400) in each group (four animals) are shown: control (a), CoPPIX (b), LPS (c), LPS+CoPPIX (d) for 24 h. Bars, 30 µm. C, Plasma aspartate aminotransferase (AST, a) and creatine kinase-MB (CK-MB, b) concentrations for untreated control, CoPPIX (5 mg/kg, 24 h), LPS (15 mg/kg for indicated times), and LPS+CoPPIX groups. Values without error bars represent a single measurement while values with error bars represent mean ± S.E. of four measurements (four animals). Statistical significance was determined between LPS and LPS+CoPPIX groups (12 h) or control and LPS groups (other time points) (**p<0.01; *p<0.05).
Figure 8.
Effects of CoPPIX on left ventricular function during LPS treatment in rats.
A, Western blot analysis of the rat heart with or without CQ treatment (20 mg/kg) with LPS treatment (15 mg/kg, 3 h) to determine the levels of LC3-II and p62. The ratios of LC3-II and p62 to actin were determined by densitometry analysis and are also represented. Each bar represents the mean ± S.E. of four animals. **p<0.01; *p<0.05 vs. none. B, Representative echocardiographs in each group are shown: control (a), LPS for 3 h (b), LPS+CoPPIX for 3 h (c), LPS for 24 h (d), LPS+CoPPIX for 24 h (e), and LPS+CQ for 24 h (f).
Table 1.
Echocardiographic parameters of left ventricular function were measured with LPS treatment (15 mg/kg) with or without CoPPIX (5 mg/kg) and CQ (20 mg/kg) for 3 and 24 h.
Figure 9.
Model of organelle turnover during LPS administration in rat heart.
TFEB pathway of autophagosomal and lysosomal biogenesis is activated in the heart to facilitate the elimination of damaged mitochondria during LPS administration. CoPPIX enhances the pathway and accelerates organelle turnover.