# Presentation: 107984

## CALCIUM ACTIVATION OF HEART MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION: ROLE OF THE Fo/F1ATPasePaul R. Territo, Vamsi K. Mootha, Stephanie A. French, and Robert S. BalabanLaboratory of Cardiac Energetics, National Heart Lung and Blood Institute, Bethesda Maryland 20892 USA

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-  Ca2+ resulted in >2.5 fold increase in NADH *F-F* slope with G/M, and a fall in  with [Ca2+], 		all without changes in ADP.O-1, indicating activation of CaDH and ANT or Fo/F1ATPase.
-  NADH *F-F* slope with SUC, increased 1.8 fold over control, with decreasing  over [Ca2+] 		indicating activation of either ANT or Fo/F1ATPase independent of CaDH.
-  Percent difference between ADP-Pi and FCCP-uncoupled respiration decreased with [Ca2+], 		verifying activation of either ANT or Fo/F1ATPase downstream of CaDH.
-  AsO4 stimulated respiration, with ANT inhibition, resulted in a marked increase in NADH, and 	decrease in  *F-F* slopes with [Ca2+], consistent with pure activation of Fo/F1ATPase.
-  Ca2+ stimulated respiration has adequate metabolic gain to support a >3 fold increase in 		mVO2 without changes in [NADH], indicating parallel activation at both CaDH and 			Fo/F1ATPase.

**Figure 3.**** **(**A**) Comparison of uncoupled and Ca2+ stimulated coupled respiration. Plot of percentage difference between State 3 and FCCP-uncoupled respiration with increasing [Ca2+]. State 3 respiration, oxidizing SUC (15mM), was initiated with ADP (500M) after Ca2+ depletion. Maximally uncoupled respiration was achieved by titration of [FCCP] (33.3nM final). Buffers and Ca2+ depletion were as indicated in methods, while [substrate] and [Ca2+] were as indicated in Figure 1 legend. Data are means  SEM, asterisks () indicate significant differences from State 3 (p0.05; dependent variable *t*-test). (**B**) Schematic diagram of metabolic uncoupling and isolation of Fo/F1ATPase with  AsO4

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**Figure 2.**** **(**A**)** **State 3 force flow slopes for mitochondria oxidizing G/M and SUC in the presence and absence of Ca2+. Buffers and Ca2+ depletion were as indicated in methods, while [substrate] and [Ca2+] were as indicated in Figure 1 legend. (**B**)** **Plot of ADP.O-1 ratios as a function of [Ca2+]. Data are means  SEM, asterisks () indicate significant differences from State 3 (p0.05; dependent variable *t*-test).

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**B**

**Figure 4.**** **(**A**)** **State 3 time course plots of mVO2 and  for mitochondria oxidizing G/M. Arrows indicate key additions of exogenous substrates, Ca2+, and ATPase. Buffers and Ca2+ depletion were as indicated in methods, while [substrate] and [Ca2+] were as indicated in Figure 1 legend. (**B**)** **Plot of NADH ratios as a function of metabolic condition. Optical spectra were obtained at steady state as indicated by blue arrows in Figure 4A. Data are means  SEM, asterisks () indicate significant differences from preceding metabolic State (p0.05; dependent variable *t*-test).

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