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Mitochondrial dehydrogenases are differentially stimulated by Ca²⁺. Ca²⁺ has also diverse regulatory effects on mitochondrial transporters and other enzymes. However, the consequences of these regulatory effects on mitochondrial oxidative phosphorylation (OxPhos) and ATP production, and the dependencies of these consequences on respiratory substrates, have not been investigated between the kidney and heart despite the fact that kidney energy requirements are second only to those of the heart. Our objective was, therefore, to elucidate these relationships in isolated mitochondria from the kidney outer medulla (OM) and heart. ADP-induced mitochondrial respiration was measured at different CaCl₂ concentrations in the presence of various respiratory substrates, including pyruvate + malate (PM), glutamate + malate (GM), alpha-ketoglutarate + malate (AM), palmitoyl-carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). The results showed that, in both heart and OM mitochondria, and for most complex I substrates, Ca²⁺ effects are biphasic: small increases in Ca²⁺ concentration stimulated, while large increases inhibited mitochondrial respiration. Furthermore, significant differences in substrate- and Ca²⁺-dependent O₂ utilization towards ATP production between heart and OM mitochondria were observed. With PM and PCM substrates, Ca²⁺ showed more prominent stimulatory effects in OM than in heart mitochondria, while with GM and AM substrates, Ca²⁺ had similar biphasic regulatory effects in both OM and heart mitochondria. In contrast, with complex II substrate SUC + ROT, only inhibitory effects on mitochondrial respiration was observed in both the heart and the OM. We conclude that the regulatory effects of Ca²⁺ on mitochondrial OxPhos and ATP synthesis are biphasic, substrate-dependent, and tissue-specific. Full article

The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP⁺C₁₀), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP⁺C₁₀ causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid β-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP⁺C₁₀ significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC. Full article