Activation of γ2-AMPK Suppresses Ribosome Biogenesis and Protects Against Myocardial Ischemia/Reperfusion Injury.

RATIONALE: AMPK (AMP-activated protein kinase) is a heterotrimeric protein that plays an important role in energy homeostasis and cardioprotection. Two isoforms of each subunit are expressed in the heart, but the isoform-specific function of AMPK remains unclear. OBJECTIVE: We sought to determine the role of γ2-AMPK in cardiac stress response using bioengineered cell lines and mouse models containing either isoform of the γ-subunit in the heart. METHODS AND RESULTS: We found that γ2 but not γ1 or γ3 subunit translocated into nucleus on AMPK activation. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylated and inactivated RNA Pol I (polymerase I)-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent downregulation of pre-rRNA level led to attenuated endoplasmic reticulum (ER) stress and cell death. Deleting γ2-AMPK led to increases in pre-rRNA level, ER stress markers, and cell death during glucose deprivation, which could be rescued by inhibition of rRNA processing or ER stress. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac-specific deletion of γ2-AMPK (cardiac knockout [cKO]). Although the total AMPK activity was unaltered in cKO hearts because of upregulation of γ1-AMPK, the lack of γ2-AMPK sensitizes the heart to myocardial ischemia/reperfusion injury. The cKO failed to suppress pre-rRNA level during ischemia/reperfusion and showed a greater infarct size. Conversely, cardiac-specific overexpression of γ2-AMPK decreased ribosome biosynthesis and ER stress during ischemia/reperfusion insult, and the infarct size was reduced. CONCLUSIONS: The γ2-AMPK translocates into the nucleus to suppress pre-rRNA transcription and ribosome biosynthesis during stress, thus ameliorating ER stress and cell death. Increased γ2-AMPK activity is required to protect against ischemia/reperfusion injury. Our study reveals an isoform-specific function of γ2-AMPK in modulating ribosome biosynthesis, cell survival, and cardioprotection.

HAX-1 regulates SERCA2a oxidation and degradation.

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1.

Nitric oxide (NO) modulates many physiological events through production of cGMP from its receptor, the NO-sensitive guanylyl cyclase (GC1). NO also appears to function in a cGMP-independent manner, via S-nitrosation (SNO), a redox-based modification of cysteine thiols. Previously, we have shown that S-nitrosated GC1 (SNO-GC1) is desensitized to NO stimulation following prolonged NO exposure or under oxidative/nitrosative stress. In animal models of nitrate tolerance and angiotensin II-induced hypertension, decreased vasodilation in response to NO correlates with GC1 thiol oxidation, but the physiological mechanism that resensitizes GC1 to NO and restores basal activity is unknown. Because GC1 interacts with the oxidoreductase protein-disulfide isomerase, we hypothesized that thioredoxin-1 (Trx1), a cytosolic oxidoreductase, could be involved in restoring GC1 basal activity and NO sensitivity because the Trx/thioredoxin reductase (TrxR) system maintains thiol redox homeostasis. Here, by manipulating activity and levels of the Trx1/TrxR system and by using a Trx1-Trap assay, we demonstrate that Trx1 modulates cGMP synthesis through an association between Trx1 and GC1 via a mixed disulfide. A proximity ligation assay confirmed the endogenous Trx1-GC1 complex in cells. Mutational analysis suggested that Cys609 in GC1 is involved in the Trx1-GC1 association and modulation of GC1 activity. Functionally, we established that Trx1 protects GC1 from S-nitrosocysteine-induced desensitization. A computational model of Trx1-GC1 interaction illustrates a possible mechanism for Trx1 to maintain basal GC1 activity and prevent/rescue GC1 desensitization to NO. The etiology of some oxidative vascular diseases may very well be explained by the dysfunction of the Trx1-GC1 association.

Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes.

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia-reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death.

Master redox regulator Trx1 upregulates SMYD1 & modulates lysine methylation.

Thioredoxin 1 (Trx1) is а antioxidant protein that regulates protein disulfide bond reduction, transnitrosylation, denitrosylation and other redox post-translational modifications. In order to better understand how Trx1 modulates downstream protective cellular signaling events following cardiac ischemia, we conducted an expression proteomics study of left ventricles (LVs) after thoracic aortic constriction stress treatment of transgenic mice with cardiac-specific over-expression of Trx1, an animal model that has been proven to withstand more stress than its non-transgenic littermates. Although previous redox post-translational modifications proteomics studies found that several cellular protein networks are regulated by Trx1-mediated disulfide reduction and transnitrosylation, we found that Trx1 regulates the expression of a limited number of proteins. Among the proteins found to be upregulated in this study was SET and MYND domain-containing protein 1 (SMYD1), a lysine methyltransferase highly expressed in cardiac and other muscle tissues and an important regulator of cardiac development. The observation of SMYD1 induction by Trx1 following thoracic aortic constriction stress is consistent with the retrograde fetal gene cardiac protection hypothesis. The results presented here suggest for the first time that, in addition to being a master redox regulator of protein disulfide bonds and nitrosation, Trx1 may also modulate lysine methylation, a non-redox post-translational modification, via the regulation of SMYD1 expression. Such crosstalk between redox signaling and a non-redox PTM regulation may provide novel insights into the functions of Trx1 that are independent from its immediate function as a protein reductase.

Identification of novel nuclear targets of human thioredoxin 1.

The dysregulation of protein oxidative post-translational modifications has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine post-translational modifications. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified because of the low Trx1 abundance in the nucleus. Here, we report the large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild-type Trx1 contains a conserved C32XXC35 motif, and the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, was sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed that might include regulating transcription, RNA processing, and nuclear pore function in human cells.

Role of NADPH oxidase in the regulation of autophagy in cardiomyocytes.

In the past several years, it has been demonstrated that the reactive oxygen species (ROS) may act as intracellular signalling molecules to activate or inhibit specific signalling pathways and regulate physiological cellular functions. It is now well-established that ROS regulate autophagy, an intracellular degradation process. However, the signalling mechanisms through which ROS modulate autophagy in a regulated manner have only been minimally clarified. NADPH oxidase (Nox) enzymes are membrane-bound enzymatic complexes responsible for the dedicated generation of ROS. Different isoforms of Nox exist with different functions. Recent studies demonstrated that Nox-derived ROS can promote autophagy, with Nox2 and Nox4 representing the isoforms of Nox implicated thus far. Nox2- and Nox4-dependent autophagy plays an important role in the elimination of pathogens by phagocytes and in the regulation of vascular- and cancer-cell survival. Interestingly, we recently found that Nox is also important for autophagy regulation in cardiomyocytes. We found that Nox4, but not Nox2, promotes the activation of autophagy and survival in cardiomyocytes in response to nutrient deprivation and ischaemia through activation of the PERK (protein kinase RNA-like endoplasmic reticulum kinase) signalling pathway. In the present paper, we discuss the importance of Nox family proteins and ROS in the regulation of autophagy, with a particular focus on the role of Nox4 in the regulation of autophagy in the heart.

Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway.

RATIONALE: Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown. OBJECTIVE: We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes. METHODS AND RESULTS: Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection. CONCLUSIONS: Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Trehalose-Induced Activation of Autophagy Improves Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Trehalose (TRE) is a natural, nonreducing disaccharide synthesized by lower organisms. TRE exhibits an extraordinary ability to protect cells against different kinds of stresses through activation of autophagy. However, the effect of TRE on the heart during stress has never been tested. OBJECTIVES: This study evaluated the effects of TRE administration in a mouse model of chronic ischemic remodeling. METHODS: Wild-type (WT) or beclin1+/- mice were subjected to permanent ligation of the left anterior descending artery (LAD) and then treated with either placebo or trehalose (1 mg/g/day intraperitoneally for 48 h, then 2% in the drinking water). After 4 weeks, echocardiographic, hemodynamic, gravimetric, histological, and biochemical analyses were conducted. RESULTS: TRE reduced left ventricular (LV) dilation and increased ventricular function in mice with LAD ligation compared with placebo. Sucrose, another nonreducing disaccharide, did not exert protective effects during post-infarction LV remodeling. Trehalose administration to mice overexpressing GFP-tagged LC3 significantly increased the number of GFP-LC3 dots, both in the presence and absence of chloroquine administration. TRE also increased cardiac LC3-II levels after 4 weeks following myocardial infarction (MI), indicating that it induced autophagy in the heart in vivo. To evaluate whether TRE exerted beneficial effects through activation of autophagy, trehalose was administered to beclin 1+/- mice. The improvement of LV function, lung congestion, cardiac remodeling, apoptosis, and fibrosis following TRE treatment observed in WT mice were all significantly blunted in beclin 1+/- mice. CONCLUSIONS: TRE reduced MI-induced cardiac remodeling and dysfunction through activation of autophagy.

Modulation of signaling mechanisms in the heart by thioredoxin 1.

Myocardial ischemia/reperfusion and heart failure are the major cardiac conditions in which an imbalance between oxidative stress and anti-oxidant mechanisms is observed. The myocardium has endogenous reducing mechanisms, including the thioredoxin (Trx) and glutathione systems, that act to scavenge reactive oxygen species (ROS) and reduce oxidized proteins. The Trx system consists of Trx, Trx reductase (TrxR), and an electron donor, NADPH, where Trx is maintained in a reduced state in the presence of TrxR and NADPH. Trx1, a major isoform of Trx, is abundantly expressed in the heart and exerts its oxidoreductase activity through conserved Cys32 and Cys35, reducing oxidized proteins through thiol disulfide exchange reactions. In this review, we will focus on molecular targets of Trx1 in the heart, including transcription factors, microRNAs, histone deactylases, and protein kinases. We will then discuss how Trx1 regulates the functions of its targets, thereby affecting the extent of myocardial injury caused by myocardial ischemia/reperfusion and the progression of heart failure.

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling.

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2- production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

mTORC2 regulates cardiac response to stress by inhibiting MST1.

The mTOR and Hippo pathways have recently emerged as the major signaling transduction cascades regulating organ size and cellular homeostasis. However, direct crosstalk between two pathways is yet to be determined. Here, we demonstrate that mTORC2 is a direct negative regulator of the MST1 kinase, a key component of the Hippo pathway. mTORC2 phosphorylates MST1 at serine 438 in the SARAH domain, thereby reducing its homodimerization and activity. We found that Rictor/mTORC2 preserves cardiac structure and function by restraining the activity of MST1 kinase. Cardiac-specific mTORC2 disruption through Rictor deletion leads to a marked activation of MST1 that, in turn, promotes cardiac dysfunction and dilation, impairing cardiac growth and adaptation in response to pressure overload. In conclusion, our study demonstrates the existence of a direct crosstalk between mTORC2 and MST1 that is critical for cardiac cell survival and growth.

The importance of autophagy in cardioprotection.

Autophagy is an intracellular lysosomal-mediated catabolic process in which senescent or damaged proteins and organelles are sequestered by double membrane-limited vesicles called autophagosomes, and then degraded by lysosomes. While the role of autophagy in different pathological states is context-dependent, it has been shown that during cardiac ischemia, autophagy is upregulated as a cardioprotective adaptation. We recently demonstrated that Rheb, a small GTP-binding protein that directly activates the complex 1 of the mechanistic target of rapamycin, is a critical regulator of autophagy during cardiac ischemia. We found that cardiac Rheb/mTORC1 signaling is activated in a deregulated manner during ischemia in obesity and metabolic syndrome. This uncontrolled activation of the Rheb/mTORC1 pathway leads to autophagy inhibition and to a reduction of myocardial tolerance to ischemia. This data further supports the relevance of autophagy as a fundamental protective mechanism during myocardial ischemia and suggests that reactivation of autophagy, in particular through the inhibition of Rheb/mTORC1 signaling may represent a promising therapeutic option to treat subjects with an acute myocardial infarction, particularly those affected by metabolic derangements. This review will deal with the biological significance of autophagy in cardioprotection.

New insights into the role of mitochondrial dynamics and autophagy during oxidative stress and aging in the heart.

The heart is highly sensitive to the aging process. In the elderly, the heart tends to become hypertrophic and fibrotic. Stiffness increases with ensuing systolic and diastolic dysfunction. Aging also affects the cardiac response to stress. At the molecular level, the aging process is associated with accumulation of damaged proteins and organelles, partially due to defects in protein quality control systems. The accumulation of dysfunctional and abnormal mitochondria is an important pathophysiological feature of the aging process, which is associated with excessive production of reactive oxygen species. Mitochondrial fusion and fission and mitochondrial autophagy are crucial mechanisms for maintaining mitochondrial function and preserving energy production. In particular, mitochondrial fission allows for selective segregation of damaged mitochondria, which are afterward eliminated by autophagy. Unfortunately, recent evidence indicates that mitochondrial dynamics and autophagy are progressively impaired over time, contributing to the aging process. This suggests that restoration of these mechanisms could delay organ senescence and prevent age-associated cardiac diseases. Here, we discuss the current understanding of the close relationship between mitochondrial dynamics, mitophagy, oxidative stress, and aging, with a particular focus on the heart.

Bone morphogenetic protein-focused strategies to induce cytotoxicity in lung cancer cells.

BACKGROUND: High bone morphogenetic protein (BMP)-2 expression in lung carcinoma correlates with poor patient prognosis. The present study explored strategies to repress BMP signaling. MATERIALS AND METHODS: The cytotoxicity of BMP2-knockdown, dorsomorphin derivatives, and microRNAs was tested in transformed and non-transformed lung cells. Microarray analyses of 1,145 microRNAs in A549 lung adenocarcinoma cells and two other transformed lung cell types relative to BEAS-2B bronchial epithelial cells were performed. RESULTS: Reduced BMP2 synthesis inhibited A549 cell growth. The dorsomorphin derivative LDN-193189, but not DMH1 or DMH4, was strongly cytotoxic towards A549 cells, but not towards BEAS-2B cells. Microarray analysis revealed that 106 miRNAs were down-regulated and 69 miRNAs were up-regulated in the three transformed lines. Three down-regulated miRNAs, hsa-mir-34b, hsa-mir-34c-3p, and hsa-miR-486-3p, repressed a BMP2 reporter gene and were cytotoxic in A549 cells, but not towards BEAS-2B cells. CONCLUSION: The observed cytotoxicity suggests that reducing BMP signaling is a useful line of attack for therapy of lung cancer.