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-mediated stimuli can modulate [Ca²⁺]_i 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 μmmol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 μmol/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 μmol/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.