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 [Ca2+](i) (fura-2 ratio), pH(i) (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop [Ca2+](i) overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of [Ca2+](i). We investigated whether cGMP-rnediatcd stimuli can modulate [Ca2+](i) or pHi recovery and whether this contributes to their protective effect. Membrane-permeable cGMP analogues, 8-bromo-cGMP (1 mmol/L) or 8-pCPT-cGMP (10 mu mmol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 mu mol/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 [Ca2+](i) 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 [Ca2+](i) 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 Ca2+ sequestration (caffeine-releasable Ca2+ pool: 2-3 fold increase vs. control). Inhibition of sarcoplasmic Ca2+-ATPase (SERCA) by thapsigargin (150 nmol/L) or of protein kinase G with KT-5823 (1 mu mol/L) abolished the effect of these stimuli on [Ca2+](i) 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 [Ca2+](i) and [Ca2+](i) oscillation during reoxygenation and to attenuate the excessive activation of the contractile machinery that otherwise leads to the development of hypercontracture. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.