Mechanism of cGMP-mediated protection in a cellular model of myocardial reperfusion injury.

OBJECTIVE

Reperfusion injury of the myocardium is characterised by development of cardiomyocyte hypercontracture. Previous studies have shown that cGMP-mediated stimuli protect against reperfusion injury, but the cellular mechanism is still unknown.

METHODS

To simulate ischemia/reperfusion, adult rat cardiomyocytes were incubated anoxically (pH(o) 6.4) and then reoxygenated (pH(o) 7.4). Cytosolic calcium Ca(2+) (fura-2 ratio), pH(i) (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop Ca(2+) overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of Ca(2+). We investigated whether cGMP-mediated stimuli can modulate Ca(2+) or pH(i) recovery and whether this contributes to their protective effect. Membrane-permeable cGMP analogues, 8-bromo-cGMP (1 mmol/L) or 8-pCPT-cGMP (10 micrommol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 micromol/L), were applied.

RESULTS

The investigated stimuli protect against reoxygenation-induced hypercontracture (cell length as percent of end-ischemic length; control: 68+/-1.6; 8-bromo-cGMP: 88+/-1.5*; 8-pCPT-cGMP: 84+/-2.9*; urodilatin: 87+/-1.1*; n=24; p<0.05). Recovery from Ca(2+) overload after 2 min reoxygenation [fura-2 ratio (a.u.); control: 1.43+/-0.15; 8-bromo-cGMP: 1.86+/-0.15; 8-pCPT-cGMP: 1.92+/-0.19*; urodilatin: 1.93+/-0.24*; n=25; p<0.05] was accelerated, and the frequency of Ca(2+) oscillations (min(-1)) was significantly reduced (control: 49+/-5.0 min(-1); 8-bromo-cGMP: 18+/-3.5 min(-1); 8-pCPT-cGMP: 18+/-4.5* min(-1); urodilatin: 16+/-4.1* min(-1); n=24; *p<0.05). cGMP-mediated stimuli increased sarcoplasmic Ca(2+) sequestration (caffeine-releasable Ca(2+) pool: 2-3 fold increase vs. control). Inhibition of sarcoplasmic Ca(2+)-ATPase (SERCA) by thapsigargin (150 nmol/L) or of protein kinase G with KT-5823 (1 micromol/L) abolished the effect of these stimuli on Ca(2+) recovery. The investigated stimuli significantly enhanced phospholamban phosphorylation.

CONCLUSIONS

We conclude that cGMP-dependent signals activate SERCA via a protein kinase G-dependent phosphorylation of phospholamban. The increase in SERCA activity seems to reduce peak Ca(2+) and Ca(2+) oscillation during reoxygenation and to attenuate the excessive activation of the contractile machinery that otherwise leads to the development of hypercontracture.