Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition

Abstract

Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca(2+) stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mm glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.

Figure 1

Cell death in cardiomyocytes submitted to 1 h SI–30 min reoxygenation A, necrotic cell death (PI⁺) when reoxygenation was performed at pH 7.4, at pH 6.4 or at pH 7.4 in the presence of 3 mm Gly. Cell death was associated with normalization of pH upon reoxygenation. Addition of Gly to the reoxygenation buffer prevented cell death associated with pH normalization. B, contribution of apoptosis (annexin⁺–PI⁻ cells) to total cell death was small and was not significantly increased by either SI or reoxygenation. C, sequestration of intracellular Ca²⁺ with BAPTA throughout the reoxygenation period did not afford any significant protection against cell death. P < 0.05 versus normoxic cells.

Figure 2

Effect of SI and reoxygenation on cardiomyocyte volume and death (PI⁺ cells) under different conditions A, cells submitted to SI experienced a significant increase in their volume, which was corrected upon reoxygenation at pH 7.4. Cell volume did not recover when Gly was present during reoxygenation or when reoxygenation was performed at pH 6.4. P < 0.05 versus normoxic cells. B, the degree of protection against death afforded by low pH closely correlated with the magnitude of the effect on cell volume. C, the degree of protection afforded by Gly was also closely correlated with its effect on cell volume.

Figure 3

Changes of intracellular pH in cardiomyocytes submitted to 1 h SI–30 min reoxygenation (Rx) A, intracellular pH immediately before SI (baseline), after 1 h SI and at the end of reoxygenation was not modified by the addition of 3 mm Gly during the reoxygenation period. B, original measurements of intracellular H⁺ concentration (a.u. of BCECF ratio fluorescence) in 2 representative experiments. Addition of Gly did not modify the time course of pH recovery during reoxygenation

Figure 4

Reoxygenation injury in isolated rat hearts A, reoxygenation-induced LDH release in control and Gly-treated hearts, and in hearts reoxygenated at pH 6.4. B, total LDH release during reoxygenation.

Figure 5

Effect of pH and Gly on mitochondrial swelling Changes in light absorbance at 520 nm in suspensions of rat heart mitochondria submitted to different experimental protocols. A, 30 min of reoxygenation (Rx) after 1 h of SI under the following conditions: at pH 7.2 (C), with 2 different respiratory substrates (5 mm succinate or 5 mm pyruvate), at pH 6.4, with 1 μm CsA or with 200 μm CaCl₂. Absorbance is expressed as the percentage value with respect to normoxic mitochondria (Nx). Both acidic reoxygenation (6.4) and CsA prevented the reoxygenation-induced fall in light absorbance, indicative of MPT. *P < 0.05 versus Nx. B, 30 min of reoxygenation (Rx) after 1 h of SI under the following conditions: at pH 7.2 (C), with 5 mm l-alanine, with 2 mm EGTA, with 3 mm Gly and with 1 μm CsA. Absorbance is expressed as the percentage value with respect to normoxic mitochondria (Nx). Addition of Gly to the reoxygenation buffer was able to completely prevent reoxygenation-induced mitochondrial swelling, as did CsA. *P < 0.05 versus Nx. C, exposure of normoxic mitochondria to 15 min of 200 μm CaCl₂ to promote MPT, with or without 3 mm Gly. Control mitochondria were not submitted to high Ca²⁺ concentration. Addition of 3 mm Gly significantly reduced swelling in Ca²⁺-stressed mitochondria. *P < 0.05 versus Control, **P < 0.05 versus 3 mm Gly. D, changes in light absorbance throughout time in fully energized mitochondria submitted to 200 μm CaCl₂ to promote MPT. The arrows indicate the time at which Ca²⁺ was added. Addition of 3 mm Gly during Ca²⁺ overload was able to prevent mitochondrial swelling more efectively than acid pH.

Figure 6

Effect of Gly on mitochondrial calcein release Calcein release from rat heart mitochondria submitted to 15 min of 200 μm CaCl₂ to promote MPT, in the presence of Gly at 2 different concentrations, in its absence, and when MPT was blocked with 0.1 μm CsA. Control group corresponds to mitochondria not submitted to Ca²⁺ overload. Gly 3 mm and 10 mm prevented calcein release, as did CsA. P < 0.05 versus control.

Figure 7

Gly content during SI–reoxygenation (Rx) Intracellular Gly content measured by NMR in cardiomyocytes submitted to 1 h SI–30 min reoxygenation in the presence (Gly⁺) or absence (Gly⁻) of Gly in the reoxygenation buffer. Control group corresponds to cells maintained under Gly-free normoxic conditions. SI–reoxygenation resulted in a reduction of intracellular Gly content while addition of 3 mm Gly to the reoxygenation buffer induced a significant increase in the intracellular Gly content. P < 0.05 versus normoxic cells, **P < 0.01 versus normoxic cells.

Figure 8

Proposed role of Gly during reperfusion injury Ischaemia and ischaemia-related conditions are associated with cytosolic derangements that favour MPT, but also with acidosis that prevents it. Reoxygenation rapidly corrects intracellular acidosis, thus allowing MPT, which is facilitated by depletion of intracellular Gly (probably through volume regulatory decrease). Treatment with Gly at the time of reoxygenation inhibits MPT despite correction of acidosis, and prevents cell death.