Mitochondrial Redox Responses To Increased Work Intensity In Rabbit Ventricular Myocytes

Abstract

Simultaneous measurement of the intrinsically fluorescent metabolic coenzymes NAD(P)H (reduced) and FAD (oxidised) enabled assessment of the mitochondrial redox response to increased work. Isolated cardiomyocytes were field stimulated and fractional shortening simultaneously recorded with epifluorescence measurements of NAD(P)H and FAD. Cells were paced at 0.5Hz and the stimulation frequency step increased to 1Hz, 2Hz and 3Hz in order to increase work intensity. NAD(P)H was excited at 340nm (fluorescence collected 455-480nm) and FAD was excited at 430nm (fluorescence collected 505-600nm).Increasing the stimulation frequency from 0.5Hz to 2Hz and 3Hz, but not 1Hz, resulted in a decrease in NAD(P)H fluorescence and an increase in FAD fluorescence, indicating oxidation of the cell environment. Reducing work intensity back to 0.5Hz pacing led to immediate recovery of metabolite fluorescence. Addition of 2mM NaCN established a completely reduced mitochondrial environment, leading to NAD(P)H fluorescence increasing to a maximum and FAD fluorescence decreasing to a minimum. Subsequent step increase in stimulation to 3Hz caused no change in NAD(P)H or FAD fluorescence. Treatment with 2μM FCCP established a completely oxidised state, resulting in NAD(P)H fluorescence falling to a minimum and FAD fluorescence increasing to a maximum. Pacing at 3Hz in this state again led to no change in metabolite fluorescence, confirming the response to increased work was mitochondrial in origin. Increasing stimulation frequency to 3Hz in the presence of the movement uncoupler cytochalasin D, minimising cell contraction, also led to no change in NAD(P)H or FAD fluorescence, thus confirming that contractile work was the cause of the change in mitochondrial redox state.In conclusion, the response to increased work intensity in cardiomyocytes is oxidation of the cell, suggesting that the mitochondria are initially unable to maintain NAD(P)H/FADH2 supply in order to cope with increased metabolic demand.