251 THE ROLE OF NITRIC OXIDE AND CALCIUM REGULATION IN CARDIOPROTECTION FROM REMOTE ISCHAEMIC PRECONDITIONING

Abstract

We have previously shown that ischaemic preconditioning (IPC) of the whole heart protects the isolated ventricular myocytes against Ca 2+ -overload injury during simulated ischaemia [1]. Nitric oxide (NO) signalling is known to modulate Ca 2+ -regulation in cardiac myocytes [2] and plays a central role in IPC [3]. We have compared the involvement NO-signalling in cardioprotection in IPC versus remote ischaemic preconditioning (rIPC). We used an isolated ventricular myocyte model of IPC of whole hearts [1] and compared this to rIPC myocytes, where naA¯ve cardiomyocytes are remotely conditioned with the superfusate from preconditioned hearts. Two models of ischaemia-reperfusion (I/R) injury were used to determine protection. 1) Ischaemia was simulated in myocytes centrifuged to a dense pellet and layered with mineral oil to prevent gaseous diffusion (37° C, 30 min), and reperfusion by dispersing the myocyte pellet into oxygenated 2mM Ca 2+ Tyrode [1] and cell death was assessed by Calcein and Propidium Iodide staining. 2) [Ca 2+ ] i was recorded from field-stimulated myocytes loaded with Fura-2 and subjected to metabolic inhibition (2 mM NaCN and 1 mM Iodoacetic acid) for 8 min followed by re-energization with 2mM Ca 2 Tyrode for 12 min. Cell injury was determined as the inability to maintain low diastolic [Ca 2+ ] i and to contract in response to electrical stimulation. Data are mean±S.E.M (n=number of experiments, hearts; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test). IPC and rIPC both significantly reduced the degree of necrotic injury compared to control myocytes [IPC 29.7±2.1% (n=32, 6; P P P P P 2+ ] i (fura-2 ratio P Our data show that the protection against necrotic cell death of rIPC involves NOS-signalling, whereas, the improved recovery of contractile function was NOS-independent. Further, rIPC was not associated with maintained Ca 2+ -regulation, as seen in true IPC.