Rat ventricular myocytes pretreated with dinitrophenol (DNP) show increased resistance to metabolic inhibition (MI) and reperfusion (Rodrigo et al. 2002). We have set out to investigate the possible role of sarcolemmal K_ATP channels and their interplay with reactive oxygen species (ROS) in functional recovery of myocytes pretreated with DNP or H₂O₂.

Ventricular myocytes were isolated enzymatically from adult rats killed by cervical dislocation, and loaded with the indicator 6-carboxy 2Ô,7Ô-dichlorodihydrocarboxyfluorescein (DCF) to measure H₂O₂ and fura-2 to measure [Ca²⁺]_i. Single channel activity was recorded from cell-attached patch electrodes (140 mM KCl); contractions were monitored with a video system and MI was achieved by superfusion with substrate-free Tyrode solution containing NaCN (2 mM) and iodoacetate (1 mM). Myocytes were pretreated with either 50 mM DNP for 5 min, or 1 mM H₂O₂ for 1 min followed by washing with substrate-free Tyrode solution for 2 min. These cells exhibited increased recovery following MI and reperfusion. 65.2 ± 4.1 % (mean ± S.E.M., n = 23; P < 0.001 vs. control, unpaired t test) of DNP-pretreated cells and 47.9 ± 1.4 % (n = 23; P < 0.001) of H₂O₂-pretreated cells recovered contractile function compared with 19.8 ± 1.9 % (n = 45) of control myocytes. Pretreated cells maintained a low resting [Ca²⁺]_i with a fura-2 ratio (340/380 nm) of 1.65 ± 0.11 (n = 122: P < 0.001) in DNP-pretreated and 2.35 ± 0.22 (n = 81; P < 0.001) in H₂O₂-pretreated cells compared with 4.05 ± 0.22 (n = 275) in control cells. During MI myocytes pretreated with either DNP or H₂O₂ developed contractile failure earlier than control myocytes at 169 ± 5 s (n = 40; P < 0.001) and 152 ± 6 s (n = 48; P < 0.001) respectively compared to 232 ± 6 (n = 61) in control cells. DNP pretreatment was also associated with early activation of sarcolemmal K_ATP channels in unstimulated myocytes, with the delay to activation significantly reduced from 6.63 ± 0.6 min (n = 5: P < 0.001) in control myocytes to 2.3 ± 0.5 min (n = 5) in DNP-pretreated myocytes. ROS production, determined as the rate of increase in relative DCF fluorescence, was increased from 0.17 ± 0.01 units min^-1 (n = 79) in normal Tyrode solution to 0.73 ± 0.05 (n = 39; P < 0.001) by 50 mM DNP.

Our results show that pretreatment with DNP or H₂O₂ increases functional recovery of myocytes after MI reperfusion and may be associated with early activation of sarcolemmal K_ATP channels during MI. We speculate that an increase in ROS may play a role in early channel activation.

This work was supported by the BHF and The Wellcome Trust.

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