Helium-Induced Changes in Circulating Caveolin in Mice Suggest a Novel Mechanism of Cardiac Protection

Figures

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  Figure 1. Langendorff perfused hearts. Helium did not result in increased recovery after global ischemia in the Langendorff perfused isolated heart 24 h after exposure. Cardiac function was measured as (A) left ventricular developed pressure (LVDP), (B) left ventricular end diastolic pressure (LVEDP), (C) contractility (dP/dtmax) and (D) lusitropy (dP/dtmin). Data are presented as mean ± standard deviation (SD) (n = 7 for helium, n = 9 for control) and results were considered significant when p < 0.05.

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  Figure 2. Cav-1 and Cav-3 levels in cardiac tissue 30 min post helium exposure. (A) Cav-1 and Cav-3 levels measured by western blot analysis in whole-heart tissue of mice as was the internal standard GAPDH, n = 8. (B) Cav-1 and Cav-3 levels measured by western blot analysis in membrane fractions of mice, as was the internal standard Na+K+-ATPase, n = 4. Data are shown as mean ± SD. No significant differences were observed in the different tissues 30 min post He exposure, suggesting that He does not have an effect on Cav-1 and Cav-3 levels in heart tissue 30 min post exposure.

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  Figure 3. Cav-1 and Cav-3 levels in different tissues at the 24-h time point. (A) Cav-1 and Cav-3 levels were significantly lower in He whole-heart tissue of helium-treated mice, measured by western blot analysis. (B) Cav-1 and Cav-3 levels were significantly lower in membrane fractions of He-treated mice, measured by western blot analysis, n = 4. (C) Cav-3 levels were significantly higher in exosomes isolated from PFP of He-treated mice, measured by western blot analysis n = 4. Data are shown as mean ± SD, p < 0.05 was considered statistically significant. As Cav-1 and Cav-3 levels are lower in whole-heart lysate and membrane fractions but higher in exosomes of PFP at the same time point, these data suggest a secretion of Cav-1 and Cav-3 from cellular membranes located in the heart into the bloodstream.

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  Figure 4. Cav-1 and Cav-3 mRNA levels. Histogram shows the results of the real-time PCR experiments of mice whole-heart tissue. Upper panel is Cav-1 over GAPDH. and lower panel is Cav-3 over GAPDH (n = 5 for control and n = 6 for helium). No significant changes were observed, suggesting that the increase of Cav-3 in exosomes from PFP at this time point is not due to higher levels of Cav-3 gene expression but through a different mechanism, such as the direct release of Cav-3 from plasma membranes.

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  Figure 5. Assessment of cardiac ultrastructure and serum membrane fluidity post helium exposure. (A) Representative TEM images from left ventricular membranes of heart cells displayed per group, magnification 11,000 ×. Caveolae are indicated by the black arrow. EC: endothelial cell, SL: sarcolemma. No ultrastructural differences in cardiomyocytes were observed. (B) The number of caveolae in the sarcolemma of left ventricular myocytes are depicted as caveolae/µm for both time points, no significant differences were observed. All groups had the same sample size (n = 4 animals/group, n = 22–33 images acquired per animal). (C) Histogram shows the results of the cholesterol assay of whole-heart tissue in µM/mL. Twenty-four hours after helium exposure significantly less cholesterol was left in whole-heart tissue, n = 6 (control), n = 8 (helium). (D) The EPR histogram shows the changes in the S order parameter 24 h after He inhalation in membrane fractions and PFP, n = 4. The S order parameter is directly proportional to the membrane rigidity. No significant changes were observed. However, a trend towards decreased rigidity in the He group and increased rigidity in PFP can be appreciated when…

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  Figure 6. Biological effects of helium-conditioned serum on mitochondrial function. All assays utilized serum collected 24 h post exposure. (A) L6 muscle cells were treated with serum (5%) from control or helium-exposed mice. Seahorse Bioscience mitochondrial stress was assessed. Oxygen consumption rate (OCR) was measured before and after the addition of inhibitors (oligomycin, a complex V inhibitor; FCCP, a protonophore; and antimycin A and rotenone, complex III and I inhibitors, respectively) to derive parameters of mitochondrial respiration. (B) After addition of serum mean OCR was calculated after normalizing for baseline OCR of the L6 cells. (C) High-resolution respirometry analysis was performed using the Oroboros O2k-Respirometer. Serum from control and helium-exposed animals was incubated with naïve mouse heart ventricular tissue. Respiratory function of complex I and II activity (CI, CII), maximum oxidative phosphorylation capacity (mOX) and maximum uncoupled capacity (mUC) are shown after addition of complex I substrates glutamate and malate. (D) Electron paramagnetic resonance (EPR) with a DEPMPO spin probe was used to assess superoxide formation in isolated mouse heart…

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  Figure 7. Complex lipid metabolites in serum. (A) Partial least squares discriminant analysis (PLS-DA) between He and control groups. (B) Heat map based on the normalized data and p values. (C) List of metabolites positively and negatively correlated with the He and control groups. (D,E) Removing the outliers determined by the Metaboanalyst outlier algorithm show the ceramide group to be differentially regulated in the He versus the control group. (F) Sphingolipid metabolic pathway associated with ceramide identified using pathway annotation.

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  Figure 8. Experimental protocol overview of organ and blood collection. C57BL/6J mice were randomized into two different groups. Subsequently, mice inhaled either 70% He or 30% oxygen for 30 min in a Plexiglas chamber. Thirty minutes or 24 h post exposure, blood was drawn and hearts were excised for further analysis. PFP: platelet free plasma, PE: post exposure.