Background:

Oxidative stress appears to be causally involved in heart failure pathogenesis, however the molecular mechanisms remain poorly elucidated and anti-oxidative therapies have not been successful, yet. Dysfunction of the mitochondrial electron transport chain is among the main sources of reactive oxygen species (ROS) in cardiomyocytes. Nitro-oleic acid (NO₂-OA) is an electrophilic molecule, which can reversibly modulate proteins by nitro-alkylation of cysteine residues thereby exerting pleiotropic, protective effects. NO₂-OA has been clinically phase 2-approved for non-cardiovascular diseases. Nitro-alkylation of the mitochondrial complex II was shown to decrease ROS release in a mouse model of ischemia and reperfusion. However, the molecular actions of NO₂-OA in the cardiomyocyte are incompletely understood. Here, we sought to identify interaction partners of NO₂-OA and characterize functional consequences in cardiomyocytes under conditions of oxidative stress.

Methods and Results:

To identify nitro-alkylated proteins in murine left ventricular tissue after treatment with NO₂-OA, we used the click-chemistry technique and biotin-streptavidin pull-down, followed by mass spectrometry. Thereby, we detected proteins involved in calcium handling, sarcomere proteins and proteins of the mitochondrial electron transport chain. Accordingly, mitochondrial respiration was analyzed in homogenates of murine left ventricular tissue. Pre-incubation with NO₂-OA (2 µM) markedly reduced complex II oxygen consumption rate and ATPase activity. Whether this effect is protective or not under conditions of heart failure of non-ischemic origin is a matter of current investigations. To further shed light on the effects of NO₂-OA on redox homeostasis, we analyzed protein carbonylation, a marker for posttranslational oxidative protein modifications. Treatment of isolated adult murine cardiomyocytes with NO₂-OA (2 μM) profoundly diminished the level of H₂O₂-induced (25 μM) protein carbonylation. We furthermore identified peroxiredoxin (PRX) to be modulated by NO₂-OA under oxidative stress, which is an important anti-oxidative protein. Interestingly, hyperoxidation (Ox) of PRX upon H₂O₂ (50 µM) treatment of cardiomyocyte cell lines was significantly decreased after pre-incubation with NO₂-OA, indicating the cysteines of the catalytic center to be blocked by nitro-alkylation.

As a further indicator of the importance of oxidative stress for cardiomyocyte function, we for the first time found that H₂O₂ (50 μM) significantly reduced sarcomere shortening in field-stimulated, isolated adult mouse cardiomyocytes, a finding that warrants further scrutiny by inhibition studies.