Naltrexone-Induced Cardiac Function Improvement is Associated With an Attenuated Inflammatory Response and Lipid Perioxidation in Volume Overloaded Rats

Figures

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  FIGURE 1. Double immunofluorescence confocal microscopy of opioid receptor mu (MOR) (A), delta (DOR) (E), and kappa (KOR) (I) (Texas red immunofluorescence) with L-type Ca2+-channel Cav1.2 (FITC green fluorescence) (B, F, J) in rat left ventricular myocardium. Note that rows of mitochondria are located between cardiomyocytes and are immunostained for MOR as indicated by the red fluorescence (arrow) (D). Nuclei identified by their bright blue using DAPI staining (D, H, L). (C–L) show the colocalization of opioid receptor subtypes mu, delta and kappa together with the L-type Ca2+-channel Cav1.2 on cardiomomyocytes of the left ventricle. Bar = 20 μm.

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  FIGURE 2. Double immunofluorescence confocal microscopy of opioid receptor subtypes mu (MOR) (A), delta (DOR) (E), and kappa (KOR) (I) (Texas red immunofluorescence) in combination with the sarcoplasmatic ryanodine receptor (RyR) (FITC green fluorescence) (B, F, J) in rat left ventricular myocardium. (C–L) show co-expression of opioid receptor subtypes MOR, DOR and KOR with RyR on the cardiomyocytes of the rat left ventricle (yellow fluorescence). Note that rows of mitochondria are located between cardiomyocytes and are immunostained for MOR (D), DOR (H) or KOR (L) as indicated by the red fluorescence (arrow). Nuclei are indicated by their bright blue fluorescence (D, H, L). Bar = 10 μm.

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  FIGURE 3. Double immunofluorescence staining of mu-opioid receptor (MOR) (A, E) (red fluorescence) and mitochondrial marker MCT02 (green fluorescence) (B, F) in rat left ventricular myocardium from controls (A–D) and rats with ACF-induced volume overload (E–H). (A–D) show that some of MOR immunoreactivity colocalized intracellularly with well-defined mitochondrial structures (mitochondrial marker MCT02) (colocalization seen as yellow immunofluorescence) (arrow). (E–H) show MOR colocalized with swollen mitochondrial structures (mitochondrial marker MTC02) of rat LV cardiomyocytes. (Bar = 10 µm).

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  FIGURE 4. Electron micrographs of the left ventricle (LV) from controls (A, B) and rats with ACF-induced volume overload (C, D). A and B display cross sections of control hearts showing intact myocardial tissue with specific cell–cell contacts and regular intercellular spaces (see *), cardiomyocytes with well-organized numerous cardiomyofibers and intact mitochondria with a preserved internal architecture. (C, D) show ultrastructure changes of the left ventricle at 28 ± 2 days after ACF induction. Note the widening of the intercellular space (see *), degenerated myofibers (arrow) and disorganized cell organelles. In addition, many mitochondria (M) showed ultrastructural abnormalities such as size enlargement with amorphous dense bodies and disruption of the internal architecture. Magnification: (A) × 10.000; (B) × 20.000, bar = 1 μm.

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  FIGURE 5. Interleukin 6 (IL-6) (A), Interleukin 12 (IL-12) (B), tumor necrosis factor α (TNF-α) (C) and malondialdehyde (MDA) (D) values of control, ACF/Vehicle- and ACF/Naltrexone-treated rats. Values of inflammatory cytokines and malondialdehyde were significantly increased in ACF/Vehicle treated rats compared to controls (IL-6: p = 0.001; IL-12: p = 0.001; TNF-alpha: p = 0.018; MDA: p = 0.005). Chronic naltrexone treatment in ACF rats was concomitant with a significant decrease in the inflammatory cytokines and lipid-peroxidation (IL-6: p = 0.047; IL-12: p = 0.035; TNF-alpha: p = 0.007; MDA: p = 0.022). Values are medians plus interquartile ranges (n = 6 rats/group). Data represent medians (range).

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  FIGURE 6. Quantitative RT-PCR Interleukin 6 (IL-6) (A), Interleukin 12 (IL-12) (B) and tumor necrosis factor α (TNF-alpha) (C) specific mRNA values of control, ACF/Vehicle- and ACF/Naltrexone-treated rats. Values of inflammatory cytokines were significantly increased in ACF/Vehicle treated rats compared to controls (IL-12: p = 0.005; TNF-alpha: p = 0.008) but not for IL-6 (p = 0.512). Chronic naltrexone treatment in ACF rats was concomitant with a significant decrease in the inflammatory cytokine mRNA (IL-12: p = 0.001; TNF-alpha: p = 0.008) but not for IL-6 (p = 0.149). Data represent medians (range) (n = 6 rats/group).