Rapid changes in NADH and flavin autofluorescence in rat cardiac trabeculae reveal large mitochondrial complex II reserve capacity.

Abstract

A photometry-based technique was developed to measure nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence and contractile properties simultaneously in intact rat trabeculae at a high time resolution. This provides insight into the function of mitochondrial complex I and II. Maximal complex I and complex II activities were determined in saponin-permeabilized right ventricular tissue by respirometry. In trabeculae, complex II function was considerably smaller than the maximal complex II activity, suggesting large complex II reserve capacity. Up-down asymmetry in NADH and FAD kinetics suggests a complex interaction between mitochondrial and contractile function. These data show that simultaneous measurement of contractile properties and NADH and FAD kinetics in cardiac trabeculae provides a mean to study the differences in complex I and II function in intact preparations in health and disease. The functional properties of cardiac mitochondria in intact preparations have been mainly studied by measurements of nicotinamide adenine dinucleotide (NADH) autofluorescence, which reflects mitochondrial complex I function. To assess complex II function, we extended this method by measuring flavin adenine dinucleotide (FAD)-related autofluorescence in electrically stimulated cardiac trabeculae isolated from the right ventricle from the rat at 27°C. NADH and FAD autofluorescence and tension responses were measured when stimulation frequency was increased from 0.5Hz to 1, 2 or 3Hz for 3min, and thereafter decreased to 0.5Hz. Maximal complex I and complex II activity in vitro were determined in saponin-permeabilized right ventricular tissue by respirometry. NADH responses upon an increase in stimulation frequency showed a rapid decline, followed by a slow recovery towards the initial level. FAD responses followed a similar time course, but in the opposite direction. The amplitudes of early rapid changes in the NADH and FAD concentration correlated well with the change in tension time integral per second (R(2) =0.833 and 0.660 for NADH and FAD, respectively), but with different slopes for the up and down transient. Maximal velocity of the increase in FAD concentration (16±4μms(-1) ), measured upon an increase in stimulation frequency from 0.5 to 3Hz was considerably smaller than that of the decrease in NADH (78±13μms(-1) ). The respiration measurements indicated that the maximal velocity of NADH utilization (143±14μms(-1) ) was 2 times smaller than that of FADH2 (291±19μms(-1) ). This indicates that in cardiac mitochondria considerable complex II activity reserve is present.