Cyclic GMP Reduces Ventricular Myocyte Stunning after Simulated Ischemia–Reperfusion

Abstract

We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia–reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10−6 M (8-Br-cGMP) and then KT 5823 10−6 M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N2–5% CO2 at 37°C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10−6 M followed by KT 5823 10−6 M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10−6 M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10−6 M decreased percentage shortening (%short) (5.0 ± 0.6 vs 3.8 ± 0.4) and the maximum velocity (Vmax, μm/s) (48.6 ± 6.9 vs 40.2 ± 6.4). KT 5823 10−6 M added after 8-Br-cGMP partially restored %short (4.6 ± 0.5) and Vmax (46.6 ± 8.0). After stunning, baseline myocytes had decreased %short (3.4 ± 0.2) and Vmax (36.0 ± 4.2). After the addition of 8-Br-cGMP, the %short (2.7 ± 0.2) and Vmax (27.6 ± 2.5) decreased further. The addition of KT 5823 did not change either the %short or the Vmax. The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 ± 0.2) and Vmax (37.2 ± 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 ± 0.4) or Vmax (29.2 ± 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase. Ischemia–reperfusion produced myocyte stunning that was reduced when 8-Br-cGMP was added prior to but not after ischemia.