We have previously shown that ischaemic preconditioning (IPC) of the whole heart protects the isolated ventricular myocytes against Ca2+-overload injury during simulated ischaemia . Nitric oxide (NO) signalling is known to modulate Ca2+-regulation in cardiac myocytes  and plays a central role in IPC . 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  and compared this to rIPC myocytes, where naÃ¯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 Ca2+ Tyrode  and cell death was assessed by Calcein and Propidium Iodide staining. 2) [Ca2+]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 Ca2 Tyrode for 12 min. Cell injury was determined as the inability to maintain low diastolic [Ca2+]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<0.001); rIPC 30.9±3.7% (n=17, 12; P<0.001); control 55.1±2.9% (n=18, 13)]. The protective effect of rIPC was abolished by the non-specific NOS inhibitor L-NAME (100 µM) at 56.5±3.8% (n=13, 10; P<0.001), when present during the rIPC stimulus and the I/R protocol. Both IPC and rIPC increased the percentage of myocytes that recovered contractile function on re-energization following metabolic inhibition, from 39.8±3.7% of control myocytes (n=30, 12), to 63.8±2.3% (n=24, 7; P<0.001) and 55.7±3.2% (n=24, 9; P<0.01) respectively. This increased recovery of contractile function was not blocked by L-NAME present during the rIPC stimulus. However, only IPC increased the percentage of cells able to maintain a low diastolic [Ca2+]i (fura-2 ratio<1) following re-energization, from 19.4±3.2% of control myocytes (n=30, 14) to 48.3±3.8% of IPC myocytes (n=24, 7; P<0.001) and 24.9±4.5% of rIPC myocytes (n=23, 9; ns).
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 Ca2+-regulation, as seen in true IPC.