In intact heart, myoglobin inactivation reduces the maximal work output and oxygen consumption. In isolated heart cells, we show a 30% reduction in respiration and decreased ATP production when myoglobin is titrimetrically inactivated. The goal of this application is to study the mechanism of myoglobin mediated oxygen transport to the mitochondria in intact isolated ventricular myocytes. The effects of specific myoglobin inactivation on selected electrical properties and ionic gradients will be studied and correlated with diminished energy reserves. An independent goal is to measure the quantitative importance of myoglobin to ATP flux in cells isolated from aged heart and test whether the maximum rate of ATP synthesis is decreased in the aged heart in part as a consequence of decreased myoglobin content. We recently described an assay for myoglobin function in freshly isolated heart cells. This model permits us to monitor simultaneously both intracellular oxygen delivery and intracellular production of ATP. We propose to use this cell system to explore the pathway of interaction of myoglobin with mitochondria to enhance oxidative phosphorylation. We will monitor the state of intracellular myoglobin spectroscopically during experiments. Intracellular calcium and pH will be monitored with fura-2 and 6- carboxyfluorescein as well as ion-selective microelectrodes, to correlate measurements in populations with measurements in individual cells. P31 NMR will be used to measure pH, and high energy phospate. The role of myoglobin function to enhance the ATP supply only becomes evident when the energy reserves of the heart are challenged beyond the capacity of intracellular compensatory mechanisms. We will manipulate the sarcoplasmic pH and calcium in myoglobin inactivated cells in order to metabolically stress the energy reserves of the cells. We use CCCP, a proton ionophore, to partially uncouple oxidative phosphorylation and simulate some of the metabolic aspects of the working heart. We observe marked change in action potential duration with the addition of CCCP and nor-epinephrine to medium bathing the cell. We will search for the mechanism underlying this effect by examining membrane conductances in voltage clamped cells. We will seek to differentiate whether gap junctional conductance is affected either by decreased total ATP production or by subsequent changes in cellular ionic gradients.
