Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity

AbstractCardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2+-depleted mitochondria (82.2±3.6% of control), in contrast to the 2-fold activation induced by 0.95μM free Ca2+, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2+ increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I+III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2+-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50μM cAMP (85±9.9%), dibutyryl-cAMP (80.1±5.2%), 8-bromo-cAMP (88.6±3.3%), or 1μM H89 (89.7±19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2+ stimulation of oxidative phosphorylation