Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation

Sascha A. Kasseckert, Inst Justus Liebig University, Germany.
Claudia Schäfer, Justus Liebig University, Germany.
Angelika Kluger, Justus Liebig University, Germany.
Dragan Gligorievski, Justus Liebig University, Germany.
Julia Tillmann, Justus Liebig University, Germany.
Klaus Dieter Schlüter, Justus Liebig University, Germany.
Thomas Noll, Justus Liebig University, Germany.
Heinrich Sauer, Justus Liebig University, Germany.
Hans Michael Piper, Justus Liebig University, Germany.
Yaser Abdallah, Aga Khan University

This work was published before the author joined Aga Khan University.

Abstract

Aims:

Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion.

The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion.

Methods and results:

Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development.

Conclusion: sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.