Nina Kaludercic

MITOCHONDRIA AND CARDIOPROTECTION

Major factors linking mitochondrial dysfunction with myocardial injury are analyzed along with protective mechanisms elicited by endogenous processes and pharmacological treatments. In particular, a reduced rate of ATP hydrolysis and a slight increase in ROS formation appear to represent the prevailing components of self-defense mechanisms, especially in the case of ischemic preconditioning. These protective processes are activated by signaling pathways, which converge on mitochondria activating the mitochondrial KATP channels and/or inhibiting the mitochondrial permeability transition pore. These pathways can also be stimulated by pharmacological treatments. Another major goal for cardioprotection is decreasing the burst in mitochondrial ROS formation that characterizes post-ischemic reperfusion. Finally, mitochondrial targets for therapeutic intervention may include the switch of substrate being utilized, because inhibition of fatty acid oxidation is associated with cardioprotective effects.

Monoamine Oxidase A–Mediated Enhanced Catabolism of Norepinephrine Contributes to Adverse Remodeling and Pump Failure in Hearts With Pressure Overload

Rationale: Monoamine oxidases (MAOs) are mitochondrial enzymes that catabolize prohypertrophic neurotransmitters, such as norepinephrine and serotonin, generating hydrogen peroxide. Because excess reactive oxygen species and catecholamines are major contributors to the pathophysiology of congestive heart failure, MAOs could play an important role in this process. Objective: Here, we investigated the role of MAO-A in maladaptive hypertrophy and heart failure. Methods and Results: We report that MAO-A activity is triggered in isolated neonatal and adult myocytes on stimulation with norepinephrine, followed by increase in cell size, reactive oxygen species production, and signs of maladaptive hypertrophy. All of these in vitro changes occur, in part, independently from &agr;- and &bgr;-adrenergic receptor–operated signaling and are inhibited by the specific MAO-A inhibitor clorgyline. In mice with left ventricular dilation and pump failure attributable to pressure overload, norepinephrine catabolism by MAO-A is increased accompanied by exacerbated oxidative stress. MAO-A inhibition prevents these changes, and also reverses fetal gene reprogramming, metalloproteinase and caspase-3 activation, as well as myocardial apoptosis. The specific role of MAO-A was further tested in mice expressing a dominant-negative MAO-A (MAO-Aneo), which were more protected against pressure overload than their wild-type littermates. Conclusions: In addition to adrenergic receptor–dependent mechanisms, enhanced MAO-A activity coupled with increased intramyocardial norepinephrine availability results in augmented reactive oxygen species generation, contributing to maladaptive remodeling and left ventricular dysfunction in hearts subjected to chronic stress.

Mitochondrial pathways for ROS formation and myocardial injury: the relevance of p66(Shc) and monoamine oxidase

Although mitochondria are considered the most relevant site for the formation of reactive oxygen species (ROS) in cardiac myocytes, a major and unsolved issue is where ROS are generated in mitochondria. Respiratory chain is generally indicated as a main site for ROS formation. However, other mitochondrial components are likely to contribute to ROS generation. Recent reports highlight the relevance of monoamine oxidases (MAO) and p66Shc. The importance of these systems in the irreversibility of ischemic heart injury will be discussed along with the cardioprotective effects elicited by both MAO inhibition and p66Shc knockout. Finally, recent evidence will be reviewed that highlight the relevance of mitochondrial ROS formation also in myocardial failure and atherosclerosis.

The cardioprotective effects elicited by p66Shc ablation demonstrate the crucial role of mitochondrial ROS formation in ischemia/reperfusion injury

Although a major contribution to myocardial ischemia-reperfusion (I/R) injury is suggested to be provided by formation of reactive oxygen species (ROS) within mitochondria, sites and mechanisms are far from being elucidated. Besides a dysfunctional respiratory chain, other mitochondrial components, such as monoamine oxidase and p66(Shc), might be involved in oxidative stress. In particular, p66(Shc) has been shown to catalyze the formation of H(2)O(2). The relationship among p66(Shc), ROS production and cardiac damage was investigated by comparing hearts from p66(Shc) knockout mice (p66(Shc-/-)) and wild-type (WT) littermates. Perfused hearts were subjected to 40 min of global ischemia followed by 15 min of reperfusion. Hearts devoid of p66(Shc) were significantly protected from I/R insult as shown by (i) reduced release of lactate dehydrogenase in the coronary effluent (25.7+/-7.49% in p66(Shc-/-) vs. 39.58+/-5.17% in WT); (ii) decreased oxidative stress as shown by a 63% decrease in malondialdehyde formation and 40+/-8% decrease in tropomyosin oxidation. The degree of protection was independent of aging. The cardioprotective efficacy associated with p66(Shc) ablation was comparable with that afforded by other antioxidant interventions and could not be increased by antioxidant co-administration suggesting that p66(Shc) is downstream of other pathways involved in ROS formation. In addition, the absence of p66(Shc) did not affect the protection afforded by ischemic preconditioning. In conclusion, the absence of p66(Shc) reduces the susceptibility to reperfusion injury by preventing oxidative stress. The present findings provide solid and direct evidence that mitochondrial ROS formation catalyzed by p66(Shc) is causally related to reperfusion damage.

Monoamine oxidases (MAO) in the pathogenesis of heart failure and ischemia/reperfusion injury

Recent evidence highlights monoamine oxidases (MAO) as another prominent source of oxidative stress. MAO are a class of enzymes located in the outer mitochondrial membrane, deputed to the oxidative breakdown of key neurotransmitters such as norepinephrine, epinephrine and dopamine, and in the process generate H(2)O(2). All these monoamines are endowed with potent modulatory effects on myocardial function. Thus, when the heart is subjected to chronic neuro-hormonal and/or peripheral hemodynamic stress, the abundance of circulating/tissue monoamines can make MAO-derived H(2)O(2) production particularly prominent. This is the case of acute cardiac damage due to ischemia/reperfusion injury or, on a more chronic stand, of the transition from compensated hypertrophy to overt ventricular dilation/pump failure. Here, we will first briefly discuss mitochondrial status and contribution to acute and chronic cardiac disorders. We will illustrate possible mechanisms by which MAO activity affects cardiac biology and function, along with a discussion as to their role as a prominent source of reactive oxygen species. Finally, we will speculate on why MAO inhibition might have a therapeutic value for treating cardiac affections of ischemic and non-ischemic origin. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Modulation of Mitochondrial Proteome and Improved Mitochondrial Function by Biventricular Pacing of Dyssynchronous Failing Hearts

Background—Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production. Methods and Results—Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-&bgr; subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, P=0.004) and specific activity (+20%, P=0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity. Conclusions—CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function.

Reactive oxygen species and redox compartmentalization.

Reactive oxygen species (ROS) formation and signaling are of major importance and regulate a number of processes in physiological conditions. A disruption in redox status regulation, however, has been associated with numerous pathological conditions. In recent years it has become increasingly clear that oxidative and reductive modifications are confined in a spatio-temporal manner. This makes ROS signaling similar to that of Ca2+ or other second messengers. Some subcellular compartments are more oxidizing (such as lysosomes or peroxisomes) whereas others are more reducing (mitochondria, nuclei). Moreover, although more reducing, mitochondria are especially susceptible to oxidation, most likely due to the high number of exposed thiols present in that compartment. Recent advances in the development of redox probes allow specific measurement of defined ROS in different cellular compartments in intact living cells or organisms. The availability of these tools now allows simultaneous spatio-temporal measurements and correlation between ROS generation and organelle and/or cellular function. The study of ROS compartmentalization and microdomains will help elucidate their role in physiology and disease. Here we will examine redox probes currently available and how ROS generation may vary between subcellular compartments. Furthermore, we will discuss ROS compartmentalization in physiological and pathological conditions focusing our attention on mitochondria, since their vulnerability to oxidative stress is likely at the basis of several diseases.

Mitochondria and vascular pathology.

Functional and structural changes in mitochondria are caused by the opening of the mitochondrial permeability transition pore (PTP) and by the mitochondrial generation of reactive oxygen species (ROS). These two processes are linked in a vicious cycle that has been extensively documented in ischemia/reperfusion injuries of the heart, and the same processes likely contribute to vascular pathology. For instance, the opening of the PTP causes cell death in isolated endothelial and vascular smooth muscle cells. Indeed, atherosclerosis is exacerbated when mitochondrial antioxidant defenses are hampered, but a decrease in mitochondrial ROS formation reduces atherogenesis. Determining the exact location of ROS generation in mitochondria is a relevant and still unanswered question. The respiratory chain is generally believed to be a main site of ROS formation. However, several other mitochondrial components likely contribute to ROS generation. Recent reports highlight the relevance of monoamine oxidases (MAO) and p66(Shc). For example, the absence of p66(Shc) in hypercholesterolemic mice has been reported to reduce the occurrence of foam cells and early atherogenic lesions. On the other hand, MAO inhibition has been shown to reduce oxidative stress in many cell types eliciting significant protection from myocardial ischemia. In conclusion, evidence will be presented to demonstrate that (i) mitochondria are major sites of ROS formation; (ii) an increase in mitochondrial ROS formation and/or a decrease in mitochondrial antioxidant defenses exacerbate atherosclerosis; and (iii) mitochondrial dysfunction is likely a relevant mechanism underlying several risk factors (i.e., diabetes, hyperlipidemia, hypertension) associated with atherosclerosis.

Mitochondrial Injury and Protection in Ischemic Pre- and Postconditioning

Mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability, yet a mild degree of mitochondrial dysfunction appears to underlie cardioprotection against injury caused by postischemic reperfusion. This review is focused on two major mechanisms of mitochondrial dysfunction, namely, oxidative stress and opening of the mitochondrial permeability transition pore. The formation of reactive oxygen species in mitochondria will be analyzed with regard to factors controlling mitochondrial permeability transition pore opening. Finally, these mitochondrial processes are analyzed with respect to cardioprotection afforded by ischemic pre- and postconditioning.

Monoamine oxidase B prompts mitochondrial and cardiac dysfunction in pressure overloaded hearts.

AIMS Monoamine oxidases (MAOs) are mitochondrial flavoenzymes responsible for neurotransmitter and biogenic amines catabolism. MAO-A contributes to heart failure progression via enhanced norepinephrine catabolism and oxidative stress. The potential pathogenetic role of the isoenzyme MAO-B in cardiac diseases is currently unknown. Moreover, it is has not been determined yet whether MAO activation can directly affect mitochondrial function. RESULTS In wild type mice, pressure overload induced by transverse aortic constriction (TAC) resulted in enhanced dopamine catabolism, left ventricular (LV) remodeling, and dysfunction. Conversely, mice lacking MAO-B (MAO-B(-/-)) subjected to TAC maintained concentric hypertrophy accompanied by extracellular signal regulated kinase (ERK)1/2 activation, and preserved LV function, both at early (3 weeks) and late stages (9 weeks). Enhanced MAO activation triggered oxidative stress, and dropped mitochondrial membrane potential in the presence of ATP synthase inhibitor oligomycin both in neonatal and adult cardiomyocytes. The MAO-B inhibitor pargyline completely offset this change, suggesting that MAO activation induces a latent mitochondrial dysfunction, causing these organelles to hydrolyze ATP. Moreover, MAO-dependent aldehyde formation due to inhibition of aldehyde dehydrogenase 2 activity also contributed to alter mitochondrial bioenergetics. INNOVATION Our study unravels a novel role for MAO-B in the pathogenesis of heart failure, showing that both MAO-driven reactive oxygen species production and impaired aldehyde metabolism affect mitochondrial function. CONCLUSION Under conditions of chronic hemodynamic stress, enhanced MAO-B activity is a major determinant of cardiac structural and functional disarrangement. Both increased oxidative stress and the accumulation of aldehyde intermediates are likely liable for these adverse morphological and mechanical changes by directly targeting mitochondria.