L-Carnitine: An Antioxidant Remedy for the Survival of Cardiomyocytes under Hyperglycemic Condition

Figures

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  Figure 1. Scheme of treatment. Upon reaching 40% confluence, H9c2 cells were treated for 48 and 72 h with 0.5 or 5 mM LC.

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  Figure 2. Attenuation of ROS production induced by H2O2 in LC-pretreated H9c2 cells. (a) Scheme of treatment: H9c2 cardiomyocytes were exposed to a stressful insult with 500 μM of H2O2 after pretreatment for 48 h with 0.5 or 5 mM LC. (b) Using the CellROX reagent assay, LC action was evaluated under oxidative stress conditions. The pretreatment with 0.5 or 5 mM LC causes a reduction in ROS production after stimulus with 500 μM of H2O2. (c) Quantification of ROS production. The quantified signal was normalized for the total nuclei number. From the DAPI images, it was possible to observe that the cells pretreated with both doses of LC show higher survival compared to the control cells. (d) Quantification of nuclei number pre- and after H2O2 injury in LC-pretreated H9c2 cells. Data are expressed as fold changes (FC) of mean ± SD. Significance: ∗∗p ≤ 0.01 vs. CONTR.

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  Figure 3. LC improves antioxidant response in H9c2 cells under hyperglycemic condition. (a) Representative Western blot and relevant quantification: LC stimuli for 48 and 72 h significantly enhanced STAT3 activation (ratio pSTAT3/STAT3). (b) Western blot analysis and relevant quantification: 5 mM LC improved SOD2 protein content in H9c2 cells after 48 and 72 h of treatment with respect to control cells. Data are expressed as fold changes (FC) of mean ± SD. Significance: ∗p ≤ 0.05 vs. CONTR; ∗∗p ≤ 0.01 vs. CONTR; §§p ≤ 0.01 vs. LC 0.5 mM.

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  Figure 4. LC downregulation of CaMKII pathway. (a) LC treatments decreased CaMKII-positive cell number and its α phosphorylated form after 72 h of stimuli: this effect was more marked for the 5 mM LC dose. The quantified signal was normalized for the total nuclei number. (b) Representative Western blot and relevant quantification: LC stimuli for 72 h promote CaMKII α isoform activation. Data are expressed as fold changes (FC) of mean ± SD. Significance: ∗p ≤ 0.05 vs. CONTR.

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  Figure 5. LC action on AMPK protein expression. Immunofluorescence assay and relevant quantification showed an increase in the number of AMPK-positive cells in LC condition at 48 and 72 h. The quantified signal was normalized for the total nuclei number. Data are expressed as fold changes (FC) of mean ± SD. Significance: ∗∗∗p ≤ 0.001 vs. CONTR; §§§p ≤ 0.001 vs. LC 5 mM.

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  Figure 6. LC action on cardiomyocyte viability. (a) Experimental scheme for growth curve and viability determination. (b) 0.5 or 5 mM LC did not alter the H9c2 proliferative potential. Viability graph shows the absence of cell mortality in all treatment conditions. (c) Phase contrast images show how treatment with 0.5 or 5 mM LC do not modify the morphology of H9c2 cells. Furthermore, there is no p21 translocation from cytoplasm to nuclei following treatment with both doses of LC. Data are expressed as fold changes (FC) of mean ± SD.