Mg2+ protects adult beating cardiomyocytes against ischaemia

Abstract

Although Mg2+ reduces infarct size in whole heart models of ischaemia/reperfusion, the cardioprotective effect of Mg2+ at the cellular level is still a controversial issue. Therefore, we tested whether Mg2+ protects cardiomyocytes against ischaemia. To accomplish this aim we used an experimental model of ischaemia that utilises single beating adult cardiomyocytes in which oxygen tension is tightly regulated without the use of oxygen scavengers or metabolic inhibitors. Taking all these into consideration, this model is probably closer to in vivo conditions than the majority of previously published cellular models of ischaemia. We found that the addition of extracellular Mg2+ (8 mM) increased the survival of cells exposed to ischaemia. As sarcolemma and mitochondria are end-effectors of cardioprotective signalling, we examined whether Mg2+ regulates sarcolemmal and mitochondrial events. Mg2+ (8 mM) did not affect the whole cell K+ current as revealed by patch clamp electrophysiology. Experiments with laser confocal microscopy and the mitochondrial membrane potential-sensitive dye, JC-1, showed that Mg2+ (8 mM) did not affect ischaemia-induced mitochondrial membrane depolarisation. However, a significantly lower JC-1 ratio was required to kill cells under control conditions than cells treated with Mg2+ (8 mM). Based on the obtained data, we conclude that Mg2+ protects single beating cardiomyocytes against ischaemia by increasing cellular resistance to the consequences of mitochondrial membrane depolarisation in the cytosol.

Figure 1

Resistance of cardiomyocytes to ischaemia in the absence (control) and presence of Mg²⁺. Original images of cardiomyocytes exposed to ischaemia under control conditions (A) and in the presence of Mg²⁺ (8 mM) (B). Lowest panel, average survival time of cardiomyocytes under conditions from A and B. Bars represent the mean ± SEM (n=5-9). *P<0.05.

Figure 2

The whole cell K⁺ current in cells under control conditions and in the presence of Mg²⁺. (A) Superimposed membrane currents evoked by identical families of voltage pulses (from -100 to 80 mV; holding potential was -40 mV) in cells under control conditions and after addition of MgSO₄ (8 mM). (B) I-V relationship for the experiment in A.

Figure 3

The effect of ischaemia on mitochondrial membrane potential in the absence (control) and presence of Mg²⁺. Original images of cardiomyocytes loaded with JC-1 in the absence (A) and presence of 8 mM MgSO₄ (B). Lowest panel, average time courses corresponding to conditions depicted in A and B. Each point represents the mean ± SEM (n=5-9).

Figure 4

The relationship between mitochondrial membrane depolarisation and cell death in the absence and presence of Mg²⁺. (A) Original images of cardiomyocytes loaded with JC-1 in the absence (control) and presence of 8 mM MgSO₄ at the moment of death. (B) Average values of the JC-1 ratio at the moment of cell death in the absence and presence of Mg²⁺. Bars represent the mean ± SEM (n=5-9). *P<0.05.