Mitochondrially targeted nitro-linoleate: a new tool for the study of cardioprotection

Cardiac ischaemia-reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO2 ), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP(+) ) moiety would enhance its protective properties.

TPP(+) -LNO2 was synthesized from aminopropyl-TPP(+) and LNO2 , and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP(+) .

TPP(+) -LNO2 protected cardiomyocytes from SIR injury more potently than the parent compound LNO2 . In addition, TPP(+) -LNO2 modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP(+) -LNO2 .

Together, these results emphasize the importance of ANT1 as a target for the protective effects of LNO2 , and suggest that TPP(+) -conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.