Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

BackgroundWe have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca2+ [Ca2+]i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca2+]i.MethodsHSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.ResultsCryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration–response curves for CaCl2 on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca2+ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl2 when tested after cryostorage. These functional effects were reflected by changes in [Ca2+]i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.ConclusionsData suggested that cryopreservation modified [Ca2+]i of venous smooth muscle cells (1) through depolarization-induced changes in Ca2+ influx and (2) through activation of Rho kinase signaling pathways.