NADH Fluorescence in Isolated Guinea-Pig and Rat Cardiomyocytes Exposed to Low or High Stimulation Rates and Effect of Metabolic Inhibition with Cyanide

Abstract

In this study we investigated whether NADH fluorescence levels changed in response to low or high rates of electrical stimulation in single ventricular myocytes isolated from rat and guinea-pig hearts, either during a single contraction or upon sustained electrical stimulation of cells. NADH levels were determined from cell autofluorescence and cell length monitored using an edge-tracking device. NADH/NAD + was obtained by addition of cyanide, 100% NADH, and carbonylcyanide- p-trifluoromethoxy phenylhydrazone (FCCP), 100% NAD +. Rat myocytes exhibited slightly higher resting fluorescence levels than guinea-pig cells; however, NADH/NAD + was higher in rat than guinea-pig cells ( P < 0.05), 24.3 ± 4.3 (N = 17) vs 14.6 ± 1.6 (N = 17), respectively. There was no change in NADH fluorescence during a single contraction when cells were stimulated at either low (0.2 Hz) or high (3 Hz) rates in either species. Furthermore, NADH levels did not change upon sustained stimulation at 3 Hz in either species. Metabolic blockade with cyanide induced a dose dependent rise in NADH fluorescence which was similar for both rat and guinea-pig myocytes and reached a maximum at ≥ 1 mM of cyanide. Although a full recovery of NADH fluorescence was seen in both types of cells after brief exposure to cyanide, the rate of recovery was significantly slower in rat myocytes; times to 90% recovery were 110 ± 29 sec, N = 6, and 264 ± 50 sec, N = 6, for guinea-pig and rat cells, respectively. This work demonstrates that although rat and guinea-pig myocytes have different resting NADH/NAD +, their response to electrical stimulation is the same, whereas in response to metabolic inhibition subtle differences are seen.