A Rho-associated kinase mitigates reperfusion-induced change in the shape of cardiac capillary endothelial cells in situ.
OBJECTIVE
We have previously demonstrated that ischaemia and reperfusion of the myocardium alter capillary dimensions and endothelial cell shape and that these changes are likely to be effected by the actomyosin contractile system in endothelial cells. Rho GTPases are involved in the regulation of cytoskeletal re-organization and in cell contraction. Rho-associated kinase regulates the sensitivity of myosin light chain to Ca(2+) in smooth muscle but not in cardiac or skeletal muscle myocytes. This study investigated the role of Rho-associated kinase in endothelial cell shape change induced by cardiac ischaemia and reperfusion. The role of Rho proteins in endothelial cell shape change in situ in the myocardial capillary bed has to date not been investigated.


METHODS
Ischaemia and reperfusion were induced in Langendorff perfused rat hearts at constant flow. Electron microscopy and immunofluorescence studies localized the beta Rho-associated kinase isotype in capillary endothelial cells. Whole capillary and luminal cross-section areas, luminal and abluminal membrane lengths were measured to monitor changes in cell dimensions. We used a ROCK inhibitor, Y-27632, to investigate the role of this protein in endothelial cell shape change.


RESULTS
ROCK1 localized primarily to intracellular membranes in endothelial cells. Morphometric analysis and a study of capillary lumen resin casts demonstrated that inhibition of the activity of this kinase with Y-27632 ablated the change in shape of endothelial cells induced by ischaemia and reperfusion.


CONCLUSIONS
These results suggest that ROCK1 is involved in cardiac capillary endothelial cell shape change in situ and that targeting the contractile system in this way may be useful in ameliorating reperfusion injury.
Glyn
Matthew C P
MC
William Harvey Research Institute, Biomedical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. m.glyn@qmw.ac.uk
Lawrenson
John G
JG
Ward
Barbara J
BJ
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002196:Capillaries; D048429:Cell Size; D016319:Corrosion Casting; D004730:Endothelium, Vascular / Q000201:enzymology / Q000648:ultrastructure*; D007150:Immunohistochemistry / Q000379:methods; D007425:Intracellular Membranes / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008854:Microscopy, Electron; D015428:Myocardial Reperfusion Injury / Q000473:pathology*; D010477:Perfusion; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D054460:rho-Associated Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; C578418:ROCK1 protein, rat; D054460:rho-Associated Kinases