Role of monoamine oxidase A in cardiac hypertrophy and transition to heart failure

Oxidative stress has been implicated in numerous pathologies and a number of intracellular sources of ROS have already been identified. Mitochondria and especially mitochondrial respiratory chain, are considered as major intracellular sources of ROS. However, other potential sites responsible for ROS generation are present in the mitochondria and could be equally important, but have not been investigated up to date. In the present thesis, we investigated the role of monoamine oxidases, flavoenzymes located at the level of outer mitochondrial membrane, in the oxidative stress in cardiac myocytes, in relation to cardiac remodeling and transition from hypertrophy to heart failure. Initially, the expression level of each MAO isoform was determined at the cardiac level. These studies showed that MAO-A is the major isoform present at the cardiac level and that low concentrations of clorgyline (0.05-1 ?M) are able to completely prevent H2O2 production in the presence of MAO substrates such as tyramine and serotonin. At this concentration clorgyline did not affect mitochondrial function or ROS production by mitochondrial respiratory chain. To investigate the role of MAO in the oxidative stress, HL-1 cardiomyocytes were treated with H2O2 or arachidonic acid to induce an increase in ROS production measured by fluorescent probe Mitotracker Red. Treatment with these agents induced a 1.6- and 1.4-fold increase in oxidative stress, respectively. When cells were pretreated with 1 ?M clorgyline, specific inhibitor of MAO-A isoform, this increase in ROS production was reduced or completely prevented. On the contrary, when cells were pretreated with specific MAO-B inhibitor selegiline, no protective effect was observed. This suggests that MAO-A is the major isoform expressed at the cardiomyocyte level and involved in the oxidative stress. To further confirm the specificity of MAO-A inhibition, we genetically silenced the expression of MAO-A by 90% by means of siRNA. Results identical to those obtained using the pharmacological inhibitor clorgyline were observed in siRNA treated cells. These results unequivocally demonstrate that MAO inhibitors are specific and that MAO-A plays an important role in the onset and amplification of oxidative stress. Given the relevant role of MAO-A in the oxidative stress, we investigated its involvement in hypertrophy and heart failure, a condition strongly favored by increased oxidative burden. In vitro studies revealed that MAO-A expression was increased by 2-fold when neonatal rat cardiomyocytes were stimulated with prohypertrophic agent norepinephrine (NE) and incubation of the cells with clorgyline reduced the extent of NE-induced hypertrophy. Furthermore, stimulation of MAO-A activity by its substrate tyramine induced the expression of NFAT3 and NFAT4, well known mediators of maladaptive hypertrophy, and this increase was significantly reduced in cells pretreated with clorgyline. These changes were paralleled by an increase in mitochondrial ROS production, which was completely prevented with clorgyline. To further confirm whether these in vitro findings could be of any significance in a more complex, in vivo setting, C57Bl6 mice were subjected to transverse aortic constriction (TAC) to induce pressure-overload. This procedure initially results in concentric hypertrophy as a compensatory mechanism for the increase in pressure, leading to eccentric hypertrophy, chamber dilation and heart failure in a long term. MAO-A expression was 3.6-fold higher in mice after 6 weeks of TAC, a time-point associated with chamber dilation and decreased left ventricular (LV) function. Inhibition of MAO-A (CLO) in these mice resulted in reduced hypertrophy and LV dimensions compared to control mice, as calculated LV mass was significantly reduced in CLO group. LV end-diastolic and endsystolic dimensions were 3.5- and 1.3-fold increased in saline treated mice, reflecting chamber remodeling and dilation. This increase in chamber dimensions was absent in CLO group. Cardiac function was also markedly improved in CLO group. Both fractional shortening and ejection fraction were comparable to the values measured in sham operated mice, while they were reduced by 50% in saline treated mice. Differences in morphological and functional data were accompanied also by changes at the molecular level. Fetal gene reprogramming, measured as increase in ANP expression was 4-fold reduced in CLO mice. Reduction in hypertrophy and improvement in cardiac function were also associated with decreased levels of oxidative stress in CLO mice, as determined by DHE staining, and reduced activation of pro-hypertrophic and pro-apoptotic pathways, determined by measuring the levels of activated Akt and cleaved caspase 3. This suggests that clorgyline exerts its protective effects by reducing the levels of oxidative stress and promoting cell viability. Taken together, these data demonstrate for the first time that MAO-A plays a major role in the onset and amplification of oxidative stress, contributing to the transition from compensated hypertrophy to dilated cardiomyopathy in vivo.