Caffeic acid ethanolamide prevents cardiac dysfunction through sirtuin dependent cardiac bioenergetics preservation

Background

Cardiac oxidative stress, bioenergetics and catecholamine play major roles in heart failure progression. However, the relationships between these three dominant heart failure factors are not fully elucidated. Caffeic acid ethanolamide (CAEA), a synthesized derivative from caffeic acid that exerted antioxidative properties, was thus applied in this study to explore its effects on the pathogenesis of heart failure.

Results

In vitro studies in HL-1 cells exposed to isoproterenol showed an increase in cellular and mitochondria oxidative stress. Two-week isoproterenol injections into mice resulted in ventricular hypertrophy, myocardial fibrosis, elevated lipid peroxidation, cardiac adenosine triphosphate and left ventricular ejection fraction decline, suggesting oxidative stress and bioenergetics changes in catecholamine-induced heart failure. CAEA restored oxygen consumption rates and adenosine triphosphate contents. In addition, CAEA alleviated isoproterenol-induced cardiac remodeling, cardiac oxidative stress, cardiac bioenergetics and function insufficiency in mice. CAEA treatment recovered sirtuin 1 and sirtuin 3 activity, and attenuated the changes of proteins, including manganese superoxide dismutase and hypoxia-inducible factor 1-α, which are the most likely mechanisms responsible for the alleviation of isoproterenol-caused cardiac injury

Conclusion

CAEA prevents catecholamine-induced cardiac damage and is therefore a possible new therapeutic approach for preventing heart failure progression.     

Mitochondrial oxidative stress mediates induction of autophagy and hypertrophy in angiotensin-II treated mouse hearts

Autophagy is characterized by recycling of cellular organelles and can be induced by several stimuli, including nutrient deprivation and oxidative stress. As a major site of free radical production during oxidative phosphorylation, mitochondria are believed to be primary targets of oxidative damage during stress. Our recent study demonstrated that angiotensin II increases cardiac mitochondrial reactive oxygen species (ROS) production, causes a decline of mitochondrial membrane potential in cardiomyocytes and increases cardiac mitochondrial protein oxidative damage and mitochondrial DNA deletions. The deleterious effects of angiotensin II on mitochondria are associated with an increase in autophagosomes and increased signaling of mitochondrial biogenesis, interpreted as an attempt to replenish the damaged mitochondria and restore energy production. Direct evidence for the central role of mitochondrial ROS was investigated by comparing the effect on mice overexpressing catalase targeted to mitochondria (mCAT) and mice overexpressing peroxisomal targeted catalase (pCAT, the natural site of catalase) challenged by angiotensin II or Gαq overexpression. The mCAT, but not pCAT, mice are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage, biogenesis and autophagy induced by angiotensin II, as well as heart failure induced by overexpression of Gαq.     

Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HepG(2) cells: mitochondria serve as the main target

The exact role of superoxide radicals (O(2)(*)(-)) in apoptosis is still a matter of debate. The main objective of the present study is to evaluate the apoptotic signalling pathway initiated by O(2)(*)(-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O(2)(*)(-)-generating system. When cells were exposed to 5 to 25 microM selenite, a temporal pattern of apoptotic events was observed following the elevation of O(2)(*)(-), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl markedly reduced O(2)(*)(-) level and suppressed the mitochondrial changes and the downstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors, was capable of attenuating O(2)(*)(-)-mediated cytochrome c release and mitochondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O(2)(*)(-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitochondria-dependent apoptotic pathway. Such knowledge may not only help to obtain a better understanding of the apoptotic effect of selenite per se, but of the role of O(2)(*)(-) in initiation and execution of apoptosis.     

Mitochondrial oxidative stress mediates induction of autophagy and hypertrophy in angiotensin-II treated mouse hearts

Autophagy is characterized by recycling of cellular organelles and can be induced by several stimuli, including nutrient deprivation and oxidative stress. As a major site of free radical production during oxidative phosphorylation, mitochondria are believed to be primary targets of oxidative damage during stress. Our recent study demonstrated that angiotensin II increases cardiac mitochondrial reactive oxygen species (ROS) production, causes a decline of mitochondrial membrane potential in cardiomyocytes and increases cardiac mitochondrial protein oxidative damage and mitochondrial DNA deletions. The deleterious effects of angiotensin II on mitochondria are associated with an increase in autophagosomes and increased signaling of mitochondrial biogenesis, interpreted as an attempt to replenish the damaged mitochondria and restore energy production. Direct evidence for the central role of mitochondrial ROS was investigated by comparing the effect on mice overexpressing catalase targeted to mitochondria (mCAT) and mice overexpressing peroxisomal targeted catalase (pCAT, the natural site of catalase) challenged by angiotensin II or Gαq overexpression. The mCAT, but not pCAT, mice are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage, biogenesis and autophagy induced by angiotensin II, as well as heart failure induced by overexpression of Gαq.     

Model mice for tissue-specific deletion of the manganese superoxide dismutase (MnSOD) gene

Manganese superoxide dismutase (MnSOD) is the enzyme that converts toxic O(2)(-) to H(2)O(2) in mitochondria. Previous reports showed that a deficiency of MnSOD in mice was neonatal lethal. Therefore, a model mouse was not available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type model mouse, we designed tissue-specific MnSOD conditional knockout mice using a Cre-loxp system. First, we crossbred MnSOD flox mice with transgenic mice expressing Cre recombinase under the control of the chicken actin promoter (CAG). We confirmed that CAG MnSOD knockout mice were completely deficient in MnSOD and died as neonates, validating the use of the Cre-loxp system. Next, we generated liver-specific MnSOD-deficient mice by crossbreeding with Alb-Cre transgenic mice. MnSOD activity and protein were both significantly downregulated in the liver of liver-specific MnSOD knockout mice. However, no obvious morphological abnormality was observed in the liver when biochemical alterations such as lipid peroxidation were not detectable, suggesting a redundant or less important physiological role for MnSOD in the liver than previously thought. In the present study, we successfully generated tissue-specific MnSOD conditional knockout mice that would provide a useful tool for the analysis of various age-associated diseases such as diabetes mellitus, Parkinson's disease, stroke, and heart disease, when crossbred with tissue-specific transgenic Cre mice.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Role of reactive oxygen species and MAPKs in vanadate-induced G(2)/M phase arrest

Cell growth arrest is an important mechanism in maintaining genomic stability and integrity in response to environmental stress. Using the human lung alveolar epithelial cancer cell line A549, we investigated the role of reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK), and p38 protein kinase in vanadate-induced cell growth arrest. Exposure of cells to vanadate led to cell growth arrest at the G(2)/M phase and caused upregulation of p21 and phospho-cdc2 and degradation of cdc25C in a time- and dose-dependent manner. Vanadate stimulated mitogen-activated protein kinases (MAPKs) family members, as determined by the phosphorylation of ERK and p38. PD98059, an inhibitor of ERK, and SB202190, an inhibitor of p38, inhibited vanadate-induced cell growth arrest, upregulation of p21 and cdc2, and degradation of cdc25C. In addition to hydroxyl radical ((*)OH) formation, cellular reduction of vanadate generated superoxide radical (O(2)(*)(-)) and hydrogen peroxide (H(2)O(2)), as determined by confocal microscopy using specific dyes. Generation of O(2)(*)(-) and H(2)O(2) was inhibited by specific antioxidant enzymes, superoxide dismutase (SOD) and catalase, respectively. ROS activate ERK and p38, which in turn upregulate p21 and cdc2 and cause degradation of cdc25C, leading to cell growth arrest at the G(2)/M phase. Specific ROS affect different MAPK family members and cell growth regulatory proteins with different potencies.     

mIGF-1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD(+) -dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.     

Pharmacogenomic profiling of an oxidative stress-mediated spongiform encephalopathy

The majority of cellular superoxide is generated in the mitochondria as a by-product of normal oxidative metabolism. In the mitochondria, superoxide is detoxified by manganese superoxide dismutase (SOD2). Mice lacking SOD2 demonstrate a multifaceted neonatal lethal phenotype, including a spongiform encephalopathy that is preventable through antioxidant treatment. The molecular events behind the observed pathology in the cortex of these mice are unknown. We hypothesized that the lack of SOD2 would result in significant changes in cortical gene expression and that therapeutically beneficial antioxidant treatment would normalize the expression of some genes, providing insight into the mechanism by which mitochondrial oxidative stress results in neurodegeneration. We report the identification of gene expression profiles associated with this paradigm, which characterize the degree of response to the pharmacologic intervention. We have identified specific pathways targeted by endogenous oxidative stress, including glutathione metabolism, iron metabolism, and cell-survival pathways centering on the kinase AKT. The normalization of expression of some of these pathways by antioxidant treatment suggests approaches to treating disease in which endogenous oxidative stress plays a role.     

IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

The role of the electron transport SDHC gene on lifespan and cancer

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. It is known that oxygen is initially converted to superoxide anion (O2-), one of reactive oxygen species (ROS), by electron leaked from mainly complex III in the electron transport system present in mitochondria, where it is the major endogenous source of ROS. We have shown that a mutation in a subunit, cytochrome b large subunit (SDHC), of complex II, also results in increasing O2- production and therefore lead to apoptosis and precocious aging in Caenorhabditis elegans. Recently, individuals with an inherited propensity for vascularized head and neck tumors (i.e., paragangliomas) have been demonstrated to contain one of several mutations in complex II. To further explore the role of oxidative stress from mitochondria on apoptosis and cancer, we established a transgenic cell line with a point mutation at the ubiquinone binding region in the SDHC gene. As expected, this mutation increased O2- production from complex II and led to excess apoptosis. Moreover, a significant fraction of the surviving cells from the apoptosis were transformed, as evidenced by increased tumor formation after injection into mice. Oxidative stress results in the damage to the cellular components including mitochondria and, therefore leads to apoptosis. Furthermore, oxidative stress must cause mutations in DNA and leads to cancer. It is suggested that oxidative stress from mitochondria play an important role of both apoptosis, which leads to precocious aging, and cancer.     

Association of mitochondrial SOD deficiency with salt-sensitive hypertension and accelerated renal senescence.

Mitochondria are the major source of superoxide (O(2)(-)) in the aerobic organisms. O(2)(-) produced by the mitochondria is converted to hydrogen peroxide by mitochondrial superoxide dismutase (SOD2). Mice with complete SOD2 deficiency (SOD2(-/-)) exhibit dilated cardiomyopathy and fatty liver leading to neonatal mortality, whereas mice with partial SOD2 deficiency (SOD2(+/-)) show evidence of O(2)(-)-induced mitochondrial damage resembling cell senescence. Since earlier studies have provided compelling evidence for the role of oxidative stress and tubulointerstitial inflammation in the pathogenesis of hypertension, we tested the hypothesis that partial SOD2 deficiency may result in hypertension. Wild-type (SOD2(+/+)) and partial SOD2-deficient (SOD2(+/-)) mice had similar blood pressures at 6-7 mo of age, but at 2 yr SOD2(+/-) mice had higher blood pressure. Oxidative stress, renal interstitial T-cell and macrophage infiltration, tubular damage, and glomerular sclerosis were all significantly increased in 2-yr-old SOD2(+/-) mice. High-salt diet induced hypertension in 6-mo-old SOD2-deficient mice but not in wild-type mice. In conclusion, partial SOD2 deficiency results in oxidative stress and renal interstitial inflammation, changes compatible with accelerated renal senescence and salt-sensitive hypertension. These findings are consistent with the pattern described in numerous other models of salt-sensitive hypertension and resemble that commonly seen in elderly humans.     

Oxidative and nitrosative stress in the maintenance of myocardial function

Reactive oxygen species (ROS) are generated by several different cellular sources, and their accumulation within the myocardium is widely considered to cause harmful oxidative stress. On the other hand, their role as second messengers has gradually emerged. The equilibrium of the nitroso/redox balance between reactive nitrogen species and ROS is crucial for the health of cardiomyocytes. This review provides a comprehensive overview of sources of oxidative stress in cardiac myocytes and describes the role of the nitroso/redox balance in cardiac pathophysiology. Although the exact mechanism of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox's) is not completely understood, Nox2 and Nox4 have particularly important roles within the myocardium. Increasing evidence suggests that Nox2 produces superoxide and Nox4 generates only hydrogen peroxide. We also discuss the key role of nitric oxide synthases (NOSs) in the maintenance of the nitroso/redox balance: uncoupled endothelial NOS has been suggested to shift from nitric oxide to ROS production, contributing to increased oxidative stress within the myocardium. Furthermore, we highlight the importance of sequentially targeting and/or regulating the specific sources of oxidative and nitrosative stress to prevent and/or reverse myocardial dysfunction. Inhibition of NADPH oxidase-dependent ROS is considered to be a potential strategy for treatment of cardiomyopathy. Neither in vivo nor clinical data are available for NADPH oxidase inhibitors. Specifically targeting the mitochondria with the antioxidant MitoQ would be a very promising translation approach, because it could prevent mitochondrial permeability transition pore opening when ROS are produced during heart reperfusion. Enhancing NO signaling could also be a promising therapeutic approach against myocardial dysfunction.     

Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential (P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production (P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by approximately 50% (P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20-30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls (P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.     

Induction of cell death in arabidopsis by superoxide in combination with salicylic acid or with protein synthesis inhibitors

Induction of programmed cell death (PCD) by oxidative stress is a widespread phenomenon in all living organisms. The degree of cell death depends on the concentration of oxidants and on environmental and physiological conditions. In plants, generation of reactive oxygen intermediates (ROI) occurs during many biotic and abiotic stresses. Recently, a number of spontaneous cell death mutants have been isolated in Arabidopsis. In one of the mutants (lsd1) induction of PCD has been attributed to superoxide (O(2)(*)(-)). Here we show that while in wild type plants generation of superoxide is symptomless, combination of O(2)(*)(-) with salicylic acid or with inhibitors of protein synthesis induced PCD. Cell death induced by these treatments was suppressed by protease inhibitors, indicating an active response. PCD induced by both treatments was preceded by nuclear condensation, which is a hallmark of apoptosis in plants and animals. These results may explain increased sensitivity to oxidative stress under certain physiological conditions, associated with high levels of salicylic acid or decrease in protein synthesis.     

Sirt1 protects the heart from aging and stress

The prevalence of heart diseases, such as coronary artery disease and congestive heart failure, increases with age. Optimal therapeutic interventions that antagonize aging may reduce the occurrence and mortality of adult heart diseases. We discuss here how molecular mechanisms mediating life span extension affect aging of the heart and its resistance to pathological insults. In particular, we review our recent findings obtained from transgenic mice with cardiac-specific overexpression of Sirt1, which demonstrated delayed aging and protection against oxidative stress in the heart. We propose that activation of known longevity mechanisms in the heart may represent a novel cardioprotection strategy against aging and certain types of cardiac stress, such as oxidative stress.