Effect of temperature on the activation of myocardial KATP channel in guinea pig ventricular myocytes: a pilot study by whole cell patch clamp recording.
BACKGROUND
The myocardial ATP sensitive potassium channel (K(ATP) channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using K(ATP) agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that K(ATP) channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial K(ATP) channel by nicorandil.


METHODS
Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4 degrees C. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IK(ATP) occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 micromol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 (Graphpad, San Diego, CA, USA). Nonlinear curve fitting was done in Clampfit (Axon) or Sigmaplot (SPSS).


RESULTS
At 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C, the time needed to open the myocardial K(ATP) channel was (81.0 +/- 0) minutes, (50.5 +/- 11.7) minutes, (28.8 +/- 2.3) minutes, (9.4 +/- 10.2) minutes and (2.3 +/- 1.0) minutes respectively (P = 0.003). The linear relationship between temperature and time needed to open the channel was y (min) = (4348.790 - 124.277x)/60, where y (min) is time needed to open K(ATP) channel, x is temperature, correlation coefficient r = -0.942 (P = 0.00), regression coefficient b = -124.277 (P = 0.00). The current densities among different temperatures were statistically different (P = 0.022), the current density was greater after the activation of K(ATP) channel at higher temperatures. The lower the temperature, the fewer cells in which K(ATP) channels could be opened. At 4 degrees C, only one cell in which the K(ATP) channel could be opened, took a quite long time (81 minutes) and the I-V curve was quite untypical.


CONCLUSIONS
K(ATP) channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open K(ATP) channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate K(ATP) channels.
Jin
San-qing
SQ
Department of Anaesthesia, Huangpu Division, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. sanqingjin@hotmail.com
Niu
Li-jun
LJ
Deng
Chun-yu
CY
Yao
Zhi-bin
ZB
Zhou
Ying-jie
YJ
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D005260:Female; D005905:Glyburide / Q000494:pharmacology; D006168:Guinea Pigs; D006352:Heart Ventricles; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism*; D020108:Nicorandil / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D013696:Temperature
D015221:Potassium Channels; D020108:Nicorandil; D000255:Adenosine Triphosphate; D005905:Glyburide