Dataset Information

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

ABSTRACT:

Background and purpose

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.

Experimental approach

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(+) .

Key results

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 .

Conclusions and implications

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.

SUBMITTER: Nadtochiy SM

PROVIDER: S-EPMC3976624 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Nadtochiy Sergiy M SM Madukwe Jerry J Hagen Fred F Brookes Paul S PS

British journal of pharmacology 20140401 8

<h4>Background and purpose</h4>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 protecti ...[more]

PMID: 24102583

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Project description:Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-? (an inhibitor of p53 transcriptional activity), pifithrin-? (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL specific pathway.

Dataset Information

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

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

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

Similar Datasets

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