Purpose: Oxidative stress plays a key role in cardiac diseases, although the sources of reactive oxygen species (ROS) have not been defined conclusively. Recent studies demonstrated that the mitochondrial enzymes monoamine oxidases (MAO) are a major source of ROS in reperfusion injury and decompensated hypertrophy. The present study characterized the molecular mechanisms responsible for the increased activity of MAO. Based upon available information, the activity of these enzymes depends mostly on substrate availability. Therefore, we aimed at identifying the major substrates of MAO in hearts undergoing oxidative stress. Mass spectrometry was used to identify and quantitate potential substrates by comparing their contents in the absence and the presence of MAO inhibition.
Methods and Results: Firstly, we applied a metabolomic profiling method to investigate changes in amine contents in isolated mouse hearts, by means of a LC-MS/MS approach in the precursor ion scanning mode. Maximal oxidative stress was induced by perfusing isolated mouse hearts with 1 mM hydrogen peroxide for 15 min. Addition of 0.5 mM pargyline to the perfusion buffer 10 min before hydrogen peroxide resulted in a significant increased content of the typical MAO substrates serotonin and epinephrine, along with histamine and its product N1-methyl histamine. N1-methyl histamine was found to be the more aboundant metabolite and its content displayed a 180% increase in pargyline-treated hearts, as compared to the untreated ones. The accumulation of MAO substrates upon pargyline treatment correlated with a reduced MAO-dependent production of hydrogen peroxide. In fact we observed a decreased extent of (i) oxidation of myofibrillar proteins, as detected by disulfide bond formation in tropomyosin (Western blot under non reducing conditions), and (ii) ROS levels in tissue, as detected by dihydroethidine (DHE) staining. Surprisingly, these findings imply that the profound injury induced by H2O2 administration is not due to a direct action. Indeed, H2O2 perfusion appears to trigger an amplification pathway whereby the increase in MAO activity due to a larger substrate availability is the end-effector of the initial oxidative stress.
Conclusions: This study provides the first information on endogenous substrates of MAO becoming available under conditions of oxidative stress that is then amplified by the increased MAO activity. The identification of histamine and N1-methyl histamine, that are involved in neurotransmission and immune response, suggests a significant trafficking of MAO substrates between myocytes and non-myocyte cells in the heart.