Triphenylphosphonium compounds preferentially inhibit long-chain fatty acid oxidation in cardiac mitochondria

Triphenylphosphonium (TPP) is a lipophilic molecule widely used in targeting compounds into the mitochondria. Despite its wide use, TPP has known mitochondrial toxicity, the characteristics of which are not completely defined. In this study, we sought to determine if the effects of TPP and TPP conjugates on mitochondrial function occur in a substrate dependent manner. To do so, we treated isolated mouse heart mitochondria with TPP, commercially available TPP derivatives MitoTEMPO and MitoSOX, and a test compound (TPP-aspirin). All TPP conjugates, except MitoTEMPO which was relatively inert, preferentially inhibited mitochondrial respiration when it was supported by palmitoyl carnitine as compared to pyruvate. This substrate selectivity was not explained by differential effects on membrane potential or electron transport chain activities, as both were largely preserved at concentrations of compounds that inhibited respiration. To identify the site of inhibition, we measured fatty acid oxidation directly and found that TPP and its conjugates significantly inhibited β-oxidation activity in energized mitochondria. Carnitine palmitoyl transferase 1 (CPT1) activity was unaffected, localizing the inhibition to the inner mitochondrial compartment. Finally, acute treatment of AC16 cells with TPP showed the same preferential inhibition of oxygen consumption rates when comparing fatty acids to pyruvate, without the loss of cell viability. Cumulatively, these results show that TPP and TPP-conjugate effects on mitochondrial function have substrate dependency by targeting fatty acid oxidation.