Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion

Abstract

The carbon dioxide/bicarbonate (CO(2)/HCO(3)(-)) pair is the main biological pH buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO(2)/HCO(3)(-) on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart, and Caenorhabditis elegans). We found that, although various concentrations of CO(2)/HCO(3)(-) do not affect function under basal conditions, ischemia-reperfusion or similar insults in the presence of higher CO(2)/HCO(3)(-) resulted in greater functional loss associated with higher oxidative damage in all models. Because the effect of CO(2)/HCO(3)(-) was observed in all models tested, we believe this buffer is an important determinant of oxidative damage after ischemia-reperfusion.

Fig. 1. Cardiac HL-1 cells present increased oxidative damage and loss of viability when submitted to IR in the presence of CO₂

Panel A: Intracellular pH was measured as described in Materials and Methods, in the presence or absence of CO₂, after 95 min stabilization. Panel B: Cell viability was measured in the absence (open bars) or presence (full bars) of 10% H₂CO₃/HCO₃⁻. Cell viability was measured as described in Materials and Methods, at 0 and 95 min, in the absence or presence of IR, as indicated. Panel C: Protein carbonyl levels were detected as described in Materials and Methods and are shown as percent of 0% CO₂ levels at 0 min. *, p < 0.05 relative to non-ischemic, 95 min; ^#, p < 0.05 relative to 95 min IR in 0% CO₂.

Fig. 2. Perfused rat hearts present increased functional loss when submitted to IR in the presence of 10% CO₂

Beats per minute (BPM, Panels A and D), left ventricular developed pressure (Panels B and E) and infarct areas (Panels C and F) were measured as described in Materials and Methods for non-ischemic (Panels A–C) or IR (Panels D–F) hearts perfused with 0% (□), 5% (♦) or 10% CO₂ (●). * p < 0.05 relative to IR with 0% CO₂

Fig. 3. Increases in CO₂ are accompanied by enhanced oxidative damage in IR hearts

The amount of carbonylated proteins (A), methionine sulfoxide (B) and nitro-tyrosine (C) was quantified as described in Materials and Methods following IR conducted under the conditions of Fig. 2. *, p < 0.05 relative to 0% CO₂; ^#, p < 0.05 relative to 5% CO₂.

Fig. 4. L-NAME does not inhibit functional loss promoted by CO₂ in IR hearts

Beats per minute (BPM, Panels A), left ventricular developed pressure (Panels B) and infarct areas (Panels C) were measured as in Fig. 2, with the addition of 200 µM L-NAME to the perfusion media. *, p < 0.05 relative to 0% CO₂.

Fig. 5. 10% CO₂ decreases touch responses in C. elegans following IR, without affecting survival

Panel A: The percentage of worms that exhibit 24-hr post-AS survival in the presence or absence of CO₂ was measured as described in Material and Methods. The N2 strain is the canonical wild type genetic background, and KWN85 contains an integrated transgene that labels mechanosensory neurons with GFP. Panel B: Proteins were extracted from living C. elegans after AS in the presence or absence of CO₂, and protein carbonyls were detected using an Oxyblot. Panel C: The response to touch stimuli of living C. elegans after AS in the presence or absence of CO₂ was measured as described in Material and Methods. *, p < 0.01 relative to 0% CO₂.

Fig. 6. 10% CO₂ increases touch neuron modifications

Touch neuron (PLML and PLMR) abnormalities such as tortuous processes and breaks (Panel A) or the accumulation of GFP aggregates in the processes (Panel B) were monitored in surviving anesthetized C. elegans after IR in the presence or absence of CO₂, as described in Material Methods. *, p < 0.05 relative to 0% CO₂.