Andrea Carpi

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.

The mitochondrial permeability transition pore and cyclophilin D in cardioprotection

Mitochondria play a central role in heart energy metabolism and Ca(2+) homeostasis and are involved in the pathogenesis of many forms of heart disease. The body of knowledge on mitochondrial pathophysiology in living cells and organs is increasing, and so is the interest in mitochondria as potential targets for cardioprotection. This critical review will focus on the permeability transition pore (PTP) and its regulation by cyclophilin (CyP) D as effectors of endogenous protective mechanisms and as potential drug targets. The complexity of the regulatory interactions underlying control of mitochondrial function in vivo is beginning to emerge, and although apparently contradictory findings still exist we believe that the network of regulatory protein interactions involving the PTP and CyPs in physiology and pathology will increase our repertoire for therapeutic interventions in heart disease. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

Background: TSPO has been proposed to be a critical regulator of the permeability transition pore (PTP). Results: TSPO-null mitochondria and cardiac tissue show no difference from controls in pore function, response to ligands, or response to ischemia/reperfusion injury. Conclusion: Regulation of the PTP by the outer membrane must rely on unknown proteins. Significance: Our results call into question studies implicating TSPO in pathological processes through the PTP. Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

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.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Ranolazine protects from doxorubicin-induced oxidative stress and cardiac dysfunction

Doxorubicin is widely used against cancer; however, it can produce heart failure (HF). Among other hallmarks, oxidative stress is a major contributor to HF pathophysiology. The late INa inhibitor ranolazine has proven effective in treating experimental HF. Since elevated [Na+]i is present in failing myocytes, and has been recently linked with reactive oxygen species (ROS) production, our aim was to assess whether ranolazine prevents doxorubicin‐induced cardiotoxicity, and whether blunted oxidative stress is a mechanism accounting for such protection.