Copyright © 2005, European Society of Cardiology
Mechanism of cGMP-mediated protection in a cellular model of myocardial reperfusion injury
Institute of Physiology, Justus-Liebig-University, Aulweg 129, D-35392 Giessen, Germany
* Corresponding author. Tel.: +49 641 99 47241; fax: +49 641 99 47239. Email address: Yaser.Abdallah{at}physiologie.med.uni-giessen.de
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 (pHo 6.4) and then reoxygenated (pHo 7.4). Cytosolic calcium [Ca2+]i (fura-2 ratio), pHi (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 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 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.
KEYWORDS Reperfusion injury; Cardiomyocytes; Calcium; cGMP; Sarcoplasmic reticulum
* Guy Vassort, Montpellier, served as guest editor for this article.
Time for primary review 21 days
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