Essential role of the Na+-Ca2+ exchanger (NCX) in glutamate-enhanced cell survival in cardiac cells exposed to hypoxia/reoxygenation

Figures

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  Figure 1. Timeline of the experimental protocols (H/R). Schematic diagram showing the H/R timeline protocol in H9c2 cells (a) and in isolated rat adult cardiomyocytes (b). Control groups were incubated under normoxic conditions at 37 °C for the entire protocol. Glutamate (1 mM)-alone or in combination with 1 µM SN-6, 300 µM DL-TBOA or 3 μg/ml oligomycin (for ATP experiments conducted in H9c2-NCX1 cells)-was administered during the reoxygenation phase. Cell viability (assessed by extracellular LDH measurement) and ROS were evaluated at the end of the reoxygenation phase in both experimental protocols. ATP content, OCR and ECAR were assessed after the first hour of reoxygenation in H9c2-NCX1 cells. CTL = control; H/R = hypoxia/reoxygenation; G = glutamate.

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  Figure 2. Effect of NCX inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2 cells (a,b) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes (d) in different experimental conditions. 1 mM glutamate, alone or in combination with 1 μM SN-6, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. Each column represents the mean ± S.E.M. of almost 5 independent experiments performed in duplicate. (a) *p < 0.001 versus CTL, CTL + G and H/R + G; #p < 0.001 versus CTL, p < 0.01 versus CTL + G and p < 0.05 versus H/R + G; §p < 0.001 versus CTL and CTL + G, p < 0.01 versus H/R + G. (b) *p < 0.01 versus CTL and CTL + G; #p < 0.001 versus CTL and p < 0.01 versus CTL + G. (d) LDH levels were normalized to the control (normoxia-exposed) group and expressed as percentage. Each column represents the mean ± S.E.M. of almost 4 independent experiments performed in duplicate. *p < 0.001 versus CTL and CTL + G and p < 0.05 versus H/R + G; #p < 0.001 versus CTL + G, p < 0.01 versus CTL and p < 0.05 versus H/R + G; §p < 0.001…

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  Figure 3. Effect of EAATs inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2-NCX1 cells (a) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes (b) in different experimental conditions. 1 mM glutamate, alone or in combination with 300 μM DL-TBOA, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. (a) Each column represents the mean ± S.E.M. of almost 6 independent experiments performed in triplicate. *p < 0.001 versus CTL and p < 0.01 versus H/R + G; #p < 0.001 versus CTL and p < 0.01 versus H/R + G; §p < 0.001 versus CTL and p < 0.01 versus H/R + G. (b) LDH levels were normalized to the control (normoxia-exposed) group and expressed as percentage. Each column represents the mean ± S.E.M. of 4 independent experiments performed in duplicate. *p < 0.001 versus control groups and p < 0.05 versus H/R + G; #p < 0.001 versus control groups and H/R + G; §p < 0.01 versus CTL and p < 0.05 versus H/R + G. CTL = control; H/R = hypoxia/reoxygenation; G = glutamate; TBOA = DL-TBOA.

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  Figure 4. Effect of NCX and EAATs inhibition on glutamate-induced ATP synthesis in H9c2-NCX1 cells subjected to H/R. (a) Intracellular ATP levels evaluated under normoxic conditions in both H9c2-WT and H9c2-NCX1 cells exposed to different glutamate concentrations (0.5 and 1 mM) for 1 h. (b,c) Intracellular ATP levels evaluated in different experimental conditions. 1 mM glutamate, alone or in combination with 1 μM SN-6 or 300 µM DL-TBOA, was added during the reoxygenation phase, then ATP levels were monitored after 1 h. ATP levels were normalized to the respective sample protein content. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. Each column represents the mean ± S.E.M. of almost 10 independent experiments performed in triplicate. (a) *p < 0.001 versus CTL and p < 0.05 versus 0.5 mM. (b) *p < 0.001 versus CTL and H/R + G; #p < 0.05 versus CTL and p < 0.001 versus H/R + G; §p < 0.01 versus CTL and p < 0.001 versus H/ + G. (c) *p < 0.001 versus CTL and H/R + G; #p < 0.001 versus CTL and H/R + G; §p < 0.001 versus CTL and H/R + G. CTL = control; H/R = hypoxia/reoxygenation; G = glutamate; TBOA = DL-TBOA.

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  Figure 5. Energy metabolism characterization in H9c2-WT and H9c2-NCX1 cells. Oxidative phosphorylation rate (a) and glycolysis rate (b) determined by Seahorse XF24 Extracellular Flux Analyzer in H9c2-WT and H9c2-NCX1 cells exposed to 3 h of hypoxia followed by 1 h of reoxygenation. Each column represents the mean ± S.E.M. of 3 replications. Where unseen, error bars overlap with the histogram outline. Differences among means were assessed by Student’s t-test. Left panels show representative traces of both OCR and ECAR experiments. Representative profiles of the Seahorse assays are also shown. (a) Maximal respiratory capacity: §P < 0.05; *p < 0.001 and #p < 0.001 versus the respective control. Spare respiratory capacity: *p < 0.001 and #p < 0.001 versus the respective control. (b) Glycolysis: *p < 0.01 versus the respective control. Glycolytic capacity: §p < 0.001 and p < 0.001 versus the respective control. Glycolytic reserve: *p < 0.01 and §p < 0.01 versus the respective control. CTL = control; H/R = hypoxia/reoxygenation.

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  Figure 6. Effect of glutamate supplementation and NCX1 inhibition on cellular metabolism in H9c2-NCX1 cells subjected to H/R challenge. Oxidative phosphorylation rate (a) and glycolysis rate (b) determined by Seahorse XF24 Extracellular Flux Analyzer in H9c2-NCX1 cells exposed to 3 h of hypoxia followed by 1 h of reoxygenation in different experimental conditions. 1 mM glutamate, alone or in combination with 1 μM SN-6, was added during the reoxygenation phase and maintained for 1 h. Each column represents the mean ± S.E.M. of 4 replications. Where unseen, error bars overlap with the histogram outline. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. Left panels show representative traces of both OCR and ECAR experiments. (a) Maximal respiratory capacity: *p < 0.001 versus CTL and H/R + G, p < 0.01 versus H/R + G + SN-6; §p < 0.001 versus all groups; #p < 0.001 versus CTL and H/R + G, p < 0.01 versus H/R. Spare respiratory capacity: *p < 0.001 versus CTL and H/R + G; §p < 0.001 versus all groups. (b) Glycolysis: *p < 0.05 versus CTL, p < 0.01 versus H/R and H/R + g + SN-6. Glycolyitic capacity: §p < 0.05 versus CTL, p < 0.001 versus H/R and H/R + G…

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  Figure 7. NCX1 and EAATs expression in H9c2-WT and H9c2-NCX1 cells subjected to H/R challenge. Quantitative densitometry showing the expression of NCX1 (a) and the Na+-dependent glutamate transporters EAAC1 (b), GLAST (c) and GLT-1 (d) in H9c2-WT and H9c2-NCX1 cells exposed to H/R. β-actin was used as loading control. Normalized optical density values are expressed as percentage of the respective control. Each column represents the mean ± S.E.M. of 3 independent experiments. Differences among means were assessed by Student’s t-test. (a) *p < 0.01 versus CTL. Representative western blot images are shown below. Full-length blots are presented in Supplementary Fig. S4. CTL = control; H/R = hypoxia/reoxygenation.

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  Figure 8. Effect of glutamate exposure on NCX activity in H9c2-NCX1 cells subjected to H/R. (a,c) Representative records of Ca2+ responses to Na+-free challenge after incubation in normoxic conditions (blu line), H/R insult (red line), and after H/R challenge in the presence of 1 mM glutamate during the entire reoxygenation phase alone (grey line) and in combination with the EAATs and NCX inhibitors (yellow line) DL-TBOA (300 µM) and SN-6 (1 µM), respectively. Fluorescence intensity was expressed as F/F0-ratio, where F is the background subtracted fluorescence intensity and F0 is the background subtracted mean fluorescence value measured from each cell at resting conditions (F/F0). (b,d) Analysis of the reverse mode of NCX1 activity in the presence of 1 mM glutamate, 300 µM DL-TBOA and 1 µM SN-6 in normoxic and hypoxic conditions. The NCX1 reverse mode activity was expressed as percentage of resting condition (∆%) after H/R protocol. For Δ% calculation, we used the maximal value of fluorescence obtained after stimulation and, as baseline, the mean of fluorescence recorded during the 30 seconds preceding the Na+-free challenge. Differences among means were assessed by one-way ANOVA followed…