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+)](i) (fura-2 ratio), pH(i) (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop [Ca(2+)](i) overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of [Ca(2+)](i). We investigated whether cGMP-mediated stimuli can modulate [Ca(2+)](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 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+)](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 [Ca(2+)](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 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+)](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 [Ca(2+)](i) and [Ca(2+)](i) oscillation during reoxygenation and to attenuate the excessive activation of the contractile machinery that otherwise leads to the development of hypercontracture.
Abdallah
Y
Y
Institute of Physiology, Justus-Liebig-University, Aulweg 129, D-35392 Giessen, Germany. Yaser.Abdallah@physiologie.med.uni-giessen.de
Gkatzoflia
A
A
Pieper
H
H
Zoga
E
E
Walther
S
S
Kasseckert
S
S
Schäfer
M
M
Schlüter
K D
KD
Piper
H M
HM
Schäfer
C
C
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D048429:Cell Size / Q000187:drug effects; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D003600:Cytosol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008856:Microscopy, Fluorescence; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D002227:Carbazoles; D007211:Indoles; C073601:KT 5823; C016276:8-bromocyclic GMP; D019284:Thapsigargin; D017869:Cyclic GMP-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D006152:Cyclic GMP; D002118:Calcium