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12 ERK activation is necessary for temperature preconditioning in isolated adult rat ventricular myocytes
  1. Y Bhagatte,
  2. N M Storey
  1. Department of Cell Physiology and Pharmacology, College of Medicine, Biological Sciences and Psychology, University of Leicester, Leicester, UK


In the present study we investigated the molecular effects of temperature preconditioning (TP) in freshly isolated adult rat cardiac myocytes. TP consisted of incubating myocytes at 16°C for 2 min, followed by 37°C for 3 min, twice. Contraction of myocytes was synchronised by electrical field stimulation at 1 Hz. Recovery of contractile function was measured following simulated ischaemia/reperfusion injury, using 7 min of perfusion with substrate-free Tyrode solution containing cyanide (2 mM) and iodoacetic acid (1 mM), followed by reperfusion with Tyrode solution for 10 min. A significant increase in contractile function after TP (52%±6 n=225, 3) was observed compared to control myocytes (29%±5 n=229, 3) (p<0.05). This protection was found to positively correlate with Ca2+ handling ability when Ca2+ levels were measured following 10 min of reperfusion. TP myocytes (132±18 nM n=67, 3) was found to significantly lower free intracellular Ca2+ concentration compared to control myocytes (301±53 nM n=69, 3) (p<0.005). In a cellular model of reperfusion injury TP was found to delay mitochondrial permeability transition pore (MPTP) opening (317±32 s n=49, 3) compared to controls (193±15 s n=44, 3) (p<0.0005). This was determined by loading myocytes with tetramethylrhodamine methyl ester (5 μM) followed by photodamage. Western blot analysis showed a significant increase of phospho- Erk in response to TP. In addition, Erk inhibitors (either U0126 (0.5 μM) or PD98059 (10 μM)) present during the TP protocol were found to abolish the TP-induced delay in MPTP opening. In conclusion, TP improved contractile function, Ca2+ handling and delayed MPTP opening. Data are presented as mean and SEM. n=myocytes, animals.

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