The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.

In mammalian cells the nuclear-encoded subunits of complex I are imported into mitochondria, where they are assembled with mt-DNA encoded subunits in the complex, or exchanged with pre-existing copies in the complex. The present work shows that in fibroblast cultures inhibition by KH7 of cAMP production in the mitochondrial matrix by soluble adenylyl cyclase (sAC) results in decreased amounts of free non-incorporated nuclear-encoded NDUFS4, NDUFV2 and NDUFA9 subunits of the catalytic moiety and inhibition of the activity of complex I. Addition of permeant 8-Br-cAMP prevents this effect of KH7. KH7 inhibits accumulation in isolated rat-liver mitochondria and incorporation in complex I of "in vitro" produced, radiolabeled NDUFS4 and NDUFV2 subunits. 8-Br-cAMP prevents also this effect of KH7. Use of protease inhibitors shows that intramitochondrial cAMP exerts this positive effect on complex I by preventing digestion of nuclear-encoded subunits by mitochondrial protease(s), whose activity is promoted by KH7 and H89, an inhibitor of PKA.

Mitochondria, responding to a wide variety of signals, including oxidative stress, are criticalin regulating apoptosis that plays a key role in the pathogenesis of a variety of cardiovasculardiseases. A number of mitochondrial proteins and pathways have been found to be involvedin the mitochondrial dependent apoptosis mechanism, such as optic atrophy 1 (OPA1), sirtuin3 (Sirt3), deacetylase enzyme and cAMP signal. In the present work we report a networkamong OPA1, Sirt3 and cAMP in ROS-dependent apoptosis. Rat myoblastic H9c2 cell lines,were treated with tert-butyl hydroperoxide (t-BHP) to induce oxidative stress-dependentapoptosis. FRET analysis revealed a selective decrease of mitochondrial cAMP in response tot-BHP treatment. This was associated with a decrease of Sirt3 protein level and proteolyticprocessing of OPA1. Pretreatment of cells with permeant analogous of cAMP (8-Br-cAMP)protected the cell from apoptosis preventing all these events. Using H89, inhibitor of theprotein kinase A (PKA), and protease inhibitors, evidences have been obtained that ROSdependentapoptosis is associated with an alteration of mitochondrial cAMP/PKA signal thatcauses degradation/proteolysis of Sirt3 that, in turn, promotes acetylation and proteolyticprocessing of OPA1.

In order to investigate whether and how a modification of mitochondrialmetabolism can affect yeast sensitivityto programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the upregulationof the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathwaytarget genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4,encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential wasmeasured, capable to consume oxygen in amanner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.