222 Augmentation of creatine kinase in vitro protects against simulated ischaemia reperfusion injury

Creatine kinase (CK) catalyses the interchange of high energy phosphates to buffer ATP levels and maintains cellular energy homeostasis. The heart expresses three isoforms: sarcomeric mitochondrial CK (CKMT2), and the cytoplasmic CKM and CKB isoforms which form homo (MM/BB)- and hetero (MB)-dimers. Impaired CK activity is associated with heart failure and increases susceptibility to ischaemia/reperfusion injury.We hypothesised that augmentation of CK isoenzymes in vitro would improve cell viability following exposure to hypoxia/reoxygenation. For this purpose we created CK overexpression systems by cloning the open reading frame of the different CK isoform sequences into pcDNA3.1 expression vector and stably selected and characterised overexpressing HEK293 cell lines.The generated cell lines displayed increased CK activity in addition to individual CK isoenzyme activities. CKMT2, CKM and CKB cells had elevated total CK activity (p