NADH supplementation decreases pinacidil-primed I K(ATP) in ventricular cardiomyocytes by increasing intracellular ATP

Abstract

1. The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I K(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36 - 37°C. Adenine nucleotides were determined by HPLC. 3. Under physiological conditions (4.3 mM ATP in the pipette solution, ATP i) I K(ATP) did not contribute to basal electrical activity. 4. The ATP-dependent potassium (K (ATP)) channel opener pinacidil activated I K(ATP) dependent on [ATP] i showing a significantly more pronounced activation at lower (1 mM) [ATP] 1. 5. Supplementation of cardiomyocytes with 300 μg ml -1 NADH (4 - 6 h) resulted in a significantly reduced I K(ATP) activation by pinacidil compared to control cells. The current density was 13.8 ± 3.78 (n = 6) versus 28.9 ± 3.38 pA pF -1 (n = 19; P < 0.05). 6. Equimolar amounts of the related compounds nicotinamide and NAD + did not achieve a similar effect like NADH. 7. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6 ± 1.88 nmol mg -1 protein versus control: 35.4 ± 2.57 nmol mg -1 protein, P < 0.000005). 8. These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I K(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.