Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC142
Conditioning of rat ventricular myocytes by serum from remotely conditioned healthy human subjects and rat hearts.
S. A. Edroos1, R. H. Ghelani1, H. E. Turrell1, N. J. Samani1, G. C. Rodrigo1
1. Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom.
Endogenous cardioprotective mechanisms that protect against ischaemia-reperfusion (I/R) injury have been identified, triggered by conditioning episodes of ischaemia, either before (preconditioning) or after (postconditioning) the index ischaemia. Remote conditioning applies the conditioning stimulus to a distant muscle bed, inducing significant cardioprotection with potential clinical relevance. We present an isolated rat myocyte I/R model to screen human serum for protection. Single ventricular myocytes were isolated by enzymatic digestion of male adult rat hearts. Ischaemia was simulated by centrifugation of isolated myocytes into a dense substrate-free pellet (37°C, 30min). Gaseous diffusion was prevented by a layer of mineral oil. Reperfusion was simulated by dispersing pellets into oxygenated Tyrode (10mM glucose, 5mM pyruvate, 37°C, 10min) followed by exposure to 1/3 hyposmotic solution. Cell viability was determined by Trypan blue staining. To simulate remote conditioning, myocytes were treated with solutions of interest either before ischaemia (rIPC), or on reperfusion (rIPostC). Two models were used; (A) Separate hearts were subjected to global I/R in three cycles of 5min to release protective factors, with collection of test solution from organ effluent on reperfusion; (B) Baseline blood samples were collected from healthy volunteers following screening to exclude hyperlipidaemia / diabetes, with ethical approval. A tourniquet was applied (30mmHg suprasytolic) to induce transient ischaemia for three cycles of 5min. Blood was then collected from the contralateral arm. Serum was isolated and stored (-80°C). I/R was compared with serum rIPC and rIPostC and necrotic injury determined using propidium iodide (PI) staining. Data are mean±SEM; one-way ANOVA, Bonferroni post-hoc test. (A) Freshly collected test solution significantly decreased Trypan blue staining (I/R 53.1±2.5%; rIPC 39.6±2.2; rIPostC 33.7±2.2; n=3, p<0.001). Freezing the test solution did not reduce protection (I/R 54.0±1.7; rIPC 39.6±1.7; rIPostC 35.7±1.3; n=3; p<0.001). (B) Conditioned serum significantly decreased PI-staining from baseline in IPC (I/R 27.5±3.1; rIPC 6.3±7.2; n=16; p<0.01) but not rIPost (24.5±6.8; ns). Subgroup analysis demonstrates a loss of rIPC protection in women>40yr in IPC (46.3±13.3; n=3) not seen in young women or men of any age. We demonstrate the release of blood borne agents on conditioning that are capable of inducing remote IPC and IPostC in rat ventricular myocytes, using two assays of cell death with differing sensitivity. We show a hitherto unrecognised absence of protection in otherwise healthy female subjects over the age of 40.
Where applicable, experiments conform with Society ethical requirements