Correction: STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

[This corrects the article DOI: 10.1371/journal.pone.0179835.].

NAPSB as a predictive marker for prognosis and therapy associated with an immuno-hot tumor microenvironment in hepatocellular carcinoma.

BACKGROUND: Napsin B Aspartic Peptidase, Pseudogene (NAPSB) was associated with CD4 + T cell infiltration in pancreatic ductal adenocarcinoma. However, the biological role of NAPSB in hepatocellular carcinoma (HCC) remains to be determined. METHODS: The expression of NAPSB in HCC as well as its clinicopathological association were analyzed using data from several public datasets. qRT-PCR was used to verify the relative expression of NAPSB in patients with HCC using the Zhongnan cohort. Kaplan-Meier analyses, and univariate and multivariate Cox regression were conducted to determine the prognosis value of NAPSB on patients with HCC. Then enrichment analyses were performed to identify the possible biological functions of NAPSB. Subsequently, the immunological characteristics of NAPSB in the HCC tumor microenvironment (TME) were demonstrated comprehensively. The role of NAPSB in predicting hot tumors and its impact on immunotherapy and chemotherapy responses was also analyzed by bioinformatics methods. RESULTS: NAPSB was downregulated in patients with HCC and high NAPSB expression showed an improved survival outcome. Enrichment analyses showed that NAPSB was related to immune activation. NAPSB was positively correlated with immunomodulators, tumor-infiltrating immune cells, T cell inflamed score and cancer-immunity cycle, and highly expressed in immuno-hot tumors. High expression of NAPSB was sensitive to immunotherapy and chemotherapy, possibly due to its association with pyroptosis, apoptosis and necrosis. CONCLUSIONS: NAPSB was correlated with an immuno-hot and inflamed TME, and tumor cell death. It can be utilized as a promising predictive marker for prognosis and therapy in HCC.

Exercise-induced late preconditioning in mice is triggered by eNOS-dependent generation of nitric oxide and activation of PKCε and is mediated by increased iNOS activity.

The purpose of this study was to assess whether short-term, mild exercise induces protection against myocardial infarction and, if so, what role the eNOS-PKCε-iNOS axis plays. Mice were subjected to 2 bouts/day of treadmill exercise (60 min at 15 m/min) for 2 consecutive days. At 24 h after the last bout of exercise, mice were subjected to a 30-min coronary artery occlusion and 24 h of reperfusion. In the exercise group (group III, wild-type mice), infarct size (25.5 ± 8.8% of risk region) was significantly (P < 0.05) reduced compared with the control groups (sham exercise, group II [63.4 ± 7.8%] and acute myocardial infarction, group I [58.6 ± 7.0%]). This effect was abolished by pretreatment with the NOS inhibitor L-NA (group VI, 56.1 ± 16.2%) and the PKC inhibitor chelerythrine (group VIII, 57.9 ± 12.5%). Moreover, the late PC effect of exercise was completely abrogated in eNOS-/- mice (group XIII, 61.0 ± 11.2%). The myocardial phosphorylated eNOS at Ser-1177 was significantly increased at 30 min after treadmill training (exercise group) compared with sham-exercised hearts. PKCε translocation was significantly increased at 30 min after exercise in WT mice but not in eNOS-/- mice. At 24 h after exercise, iNOS protein was upregulated compared with sham-exercised hearts. The protection of late PC was abrogated in iNOS-/- mice (group XVI, 56.4 ± 12.9%) and in wildtype mice given the selective iNOS inhibitor 1400 W prior to ischemia (group X 62.0 ± 8.8% of risk region). We conclude that 1) even short, mild exercise induces a delayed PC effect that affords powerful protection against infarction; 2) this cardioprotective effect is dependent on activation of eNOS, eNOS-derived NO generation, and subsequent PKCε activation during PC; 3) the translocation of PKCε is dependent on eNOS; 4) the protection 24 h later is dependent on iNOS activity. Thus, eNOS is the trigger and iNOS the mediator of PC induced by mild exercise.

Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and ß-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and ß-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.

Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction.

RATIONALE: The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. OBJECTIVE: To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy and to elucidate the underlying molecular pathways. METHODS AND RESULTS: Wild-type and IL-6 knockout (IL-6(-/-)) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6(-/-) mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6(-/-) hearts after TAC. Transcriptional and protein assays of myocardial tissue identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 3 (STAT3) activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from wild-type and IL-6(-/-) mice exposed to prohypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together, these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. CONCLUSIONS: Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension.

Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury.

Acute lung injury (ALI) during sepsis is characterized by bilateral alveolar infiltrates, lung edema and respiratory failure. Here, we examined the efficacy the DNA methyl transferase (DNMT) inhibitor 5-Aza 2-deoxycytidine (Aza), the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), as well as the combination therapy of Aza and TSA (Aza+TSA) provides in the protection of ALI. In LPS-induced mouse ALI, post-treatment with a single dose of Aza+TSA showed substantial attenuation of adverse lung histopathological changes and inflammation. Importantly, these protective effects were due to substantial macrophage phenotypic changes observed in LPS-stimulated macrophages treated with Aza+TSA as compared with untreated LPS-induced macrophages or LPS-stimulated macrophages treated with either drug alone. Further, we observed significantly lower levels of pro-inflammatory molecules and higher levels of anti-inflammatory molecules in LPS-induced macrophages treated with Aza+TSA than in LPS-induced macrophages treated with either drug alone. The protection was ascribed to dual effects by an inhibition of MAPK-HuR-TNF and activation of STAT3-Bcl2 pathways. Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis.

Endoplasmic reticulum stress-dependent activation of ATF3 mediates the late phase of ischemic preconditioning.

Ischemic preconditioning (PC) is an adaptive response to transient myocardial ischemia that protects the heart from subsequent ischemia/reperfusion (I/R) injury. However, the mechanisms underlying its cardioprotective effects remain unclear. Myocardium of adult male C57/BL6 mice, preconditioned by 6 cycles of 4 minute coronary occlusion and reperfusion, showed nuclear translocation of ATF3 and ATF6 and PERK phosphorylation 30 min after PC. The abundance of ER proteins, ATF3 and ATF4 was increased 24h after PC; however, there was no evidence of IRE-1 activation in WT or ER-stress activated indicator (ERAI) mice expressing XBP-1-Venus fusion protein. PC-induced nuclear translocation of ATF3 was attenuated in transgenic mice with cardiac-restricted overexpression of inducible ATF6. Ischemic PC increased the abundance of inducible nitric oxide synthase, cyclooxygenase-2, heme oxygenase-1 and aldose reductase to levels similar between WT and ATF3-null hearts; however, the increase in IL-6 and ICAM-1 was exaggerated in ATF3-null hearts. Genetic deletion of ATF3 did not increase infarct size in non-preconditioned hearts but abolished the cardioprotective effects of PC. Larger infarct size in preconditioned ATF3-null hearts was associated with greater neutrophil infiltration in the myocardium, but no ATF3-dependent changes in the total or relative abundance of inflammatory monocytes were observed. Ischemic PC activates the unfolded protein response (UPR) and the activation of ATF3 by ER stress is essential for the cardioprotective effects of late PC.

Combinatorial therapy with acetylation and methylation modifiers attenuates lung vascular hyperpermeability in endotoxemia-induced mouse inflammatory lung injury.

Impairment of tissue fluid homeostasis and migration of inflammatory cells across the vascular endothelial barrier are crucial factors in the pathogenesis of acute lung injury (ALI). The goal for treatment of ALI is to target pathways that lead to profound dysregulation of the lung endothelial barrier. Although studies have shown that chemical epigenetic modifiers can limit lung inflammation in experimental ALI models, studies to date have not examined efficacy of a combination of DNA methyl transferase inhibitor 5-Aza 2-deoxycytidine and histone deacetylase inhibitor trichostatin A (herein referred to as Aza+TSA) after endotoxemia-induced mouse lung injury. We tested the hypothesis that treatment with Aza+TSA after lipopolysaccharide induction of ALI through epigenetic modification of lung endothelial cells prevents inflammatory lung injury. Combinatorial treatment with Aza+TSA mitigated the increased endothelial permeability response after lipopolysaccharide challenge. In addition, we observed reduced lung inflammation and lung injury. Aza+TSA also significantly reduced mortality in the ALI model. The protection was ascribed to inhibition of the eNOS-Cav1-MLC2 signaling pathway and enhanced acetylation of histone markers on the vascular endothelial-cadherin promoter. In summary, these data show for the first time the efficacy of combinatorial Aza+TSA therapy in preventing ALI in lipopolysaccharide-induced endotoxemia and raise the possibility of an essential role of DNA methyl transferase and histone deacetylase in the mechanism of ALI.

Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium.

A growing body of evidence indicates that carbon monoxide (CO), once perceived merely as a poisonous gas, exerts antiapoptotic and cytoprotective effects. Using a water-soluble CO-releasing molecule (CORM) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), we previously reported that CO induces a delayed protection against myocardial infarction similar to that observed in the late phase of ischemic preconditioning (PC). In the current study, we investigated the molecular mechanisms underlying this cardioprotective effect. The impact on apoptotic signaling pathways was first examined in the setting of ischemia/reperfusion injury. Mice were pretreated with CORM-3 or iCORM-3 (which does not release CO) and subjected to coronary occlusion/reperfusion 24h later. In mice that received CORM-3, there was a significant reduction in markers of apoptosis (cleaved lamin A, cleaved caspase-3, and cleaved PARP-1) after ischemia/reperfusion injury. To elucidate the mechanism of CORM-3-induced cardioprotection we further examined the activation of transcription factors and induction of cardioprotective and apoptosis modulating proteins. Infusion of CORM-3 rapidly activated the stress-responsive transcription factors nuclear factor kappaB (NF-κB), signal transducers and activators of transcription (STAT)1, STAT3, and NF-E2-related factor-2 (Nrf2). This was followed 24h later by upregulation of cardioprotective proteins (heme oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and extracellular superoxide dismutase [Ec-SOD]) and antiapoptotic proteins involving both the mitochondria-mediated (Mcl-1) and the death receptor-mediated (c-FLIP(S) and c-FLIP(L)) apoptosis pathways. We conclude that CO released by CORM-3 triggers a cardioprotective signaling cascade that recruits the transcription factors NF-κB, STAT1/3, and Nrf2 with a subsequent increase in cardioprotective and antiapoptotic molecules in the myocardium leading to the late PC-mimetic infarct-sparing effects. This article is part of a Special Issue entitled 'Possible Editorial'.

Hematopoietic cytokines for cardiac repair: mobilization of bone marrow cells and beyond.

Hematopoietic cytokines, traditionally known to influence cellular proliferation, differentiation, maturation, and lineage commitment in the bone marrow, include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, stem cell factor, Flt-3 ligand, and erythropoietin among others. Emerging evidence suggests that these cytokines also exert multifarious biological effects on diverse nonhematopoietic organs and tissues. Although the precise mechanisms remain unclear, numerous studies in animal models of myocardial infarction (MI) and heart failure indicate that hematopoietic cytokines confer potent cardiovascular benefits, possibly through mobilization and subsequent homing of bone marrow-derived cells into the infarcted heart with consequent induction of myocardial repair involving multifarious mechanisms. In addition, these cytokines are also known to exert direct cytoprotective effects. However, results from small-scale clinical trials of G-CSF therapy as a single agent after acute MI have been discordant and largely disappointing. It is likely that cardiac repair following cytokine therapy depends on a number of known and unknown variables, and further experimental and clinical studies are certainly warranted to accurately determine the true therapeutic potential of such therapy. In this review, we discuss the biological features of several key hematopoietic cytokines and present the basic and clinical evidence pertaining to cardiac repair with hematopoietic cytokine therapy.

A murine model of inducible, cardiac-specific deletion of STAT3: its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning.

Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~1 month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse (MCM TG:STAT3(flox/flox)) by interbreeding STAT3(flox/flox) mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG:STAT3(flox/flox) mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG:STAT3(flox/flox) mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-x(L), c-FLIP(L), c-FLIP(S)) proteins 24h later; in contrast, in tamoxifen-treated MCM TG:STAT3(flox/flox) mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35 days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIP(L) and c-FLIP(S)) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-x(L)).

O-linked ß-N-acetylglucosamine transferase is indispensable in the failing heart.

The failing heart is subject to elevated metabolic demands, adverse remodeling, chronic apoptosis, and ventricular dysfunction. The interplay among such pathologic changes is largely unknown. Several laboratories have identified a unique posttranslational modification that may have significant effects on cardiovascular function. The O-linked ß-N-acetylglucosamine (O-GlcNAc) posttranslational modification (O-GlcNAcylation) integrates glucose metabolism with intracellular protein activity and localization. Because O-GlcNAc is derived from glucose, we hypothesized that altered O-GlcNAcylation would occur during heart failure and figure prominently in its pathophysiology. After 5 d of coronary ligation in WT mice, cardiac O-GlcNAc transferase (OGT; which adds O-GlcNAc to proteins) and levels of O-GlcNAcylation were significantly (P < 0.05) elevated in the surviving remote myocardium. We used inducible, cardiac myocyte-specific Cre recombinase transgenic mice crossed with loxP-flanked OGT mice to genetically delete cardiomyocyte OGT (cmOGT KO) and ascertain its role in the failing heart. After tamoxifen induction, cardiac O-GlcNAcylation of proteins and OGT levels were significantly reduced compared with WT, but not in other tissues. WT and cardiomyocyte OGT KO mice underwent nonreperfused coronary ligation and were followed for 4 wk. Although OGT deletion caused no functional change in sham-operated mice, OGT deletion in infarcted mice significantly exacerbated cardiac dysfunction compared with WT. These data provide keen insights into the pathophysiology of the failing heart and illuminate a previously unrecognized point of integration between metabolism and cardiac function in the failing heart.

Divergent tumor necrosis factor receptor-related remodeling responses in heart failure: role of nuclear factor-kappaB and inflammatory activation.

BACKGROUND: Although preclinical data suggested that tumor necrosis factor-alpha (TNF) neutralization in heart failure (HF) would be beneficial, clinical trials of TNF antagonists were paradoxically negative. We hypothesized that TNF induces opposing inflammatory and remodeling responses in HF that are TNF-receptor (TNFR) specific. METHODS AND RESULTS: HF was induced in wild-type (WT), TNFR1(-/-), and TNFR2(-/-) mice via coronary ligation. Compared with WT HF, 4-week postinfarction survival was significantly improved in both TNFR1(-/-) and TNFR2(-/-) HF. Compared with sham, WT HF hearts exhibited significant remodeling with robust activation of nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase, and JNK2 and upregulation of TNF, interleukin (IL)-1beta, IL-6, and IL-10. Compared with WT HF, TNFR1(-/-) HF exhibited (1) improved remodeling, hypertrophy, and contractile function; (2) less apoptosis; and (3) diminished NF-kappaB, p38 mitogen-activated protein kinase, and JNK2 activation and cytokine expression. In contrast, TNFR2(-/-) HF showed exaggerated remodeling and hypertrophy, increased border zone fibrosis, augmented NF-kappaB and p38 mitogen-activated protein kinase activation, higher IL-1beta and IL-6 gene expression, greater activated macrophages, and greater apoptosis. Oxidative stress and diastolic function were improved in both TNFR1(-/-)and TNFR2(-/-) HF. In H9c2 cardiomyocytes, sustained NF-kappaB activation was proapoptotic, an effect dependent on TNFR1 signaling, whereas TNFR2 overexpression attenuated TNF-induced NF-kappaB activation. CONCLUSIONS: TNFR1 and TNFR2 have disparate and opposing effects on remodeling, hypertrophy, NF-kappaB, inflammation, and apoptosis in HF: TNFR1 exacerbates, whereas TNFR2 ameliorates, these events. However, signaling through both receptors is required to induce diastolic dysfunction and oxidative stress. TNFR-specific effects in HF should be considered when therapeutic anti-TNF strategies are developed.

Cardiac myocyte-specific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury. METHODS AND RESULTS: Constitutive overexpression of iNOS in transgenic mice (alpha-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[(3)H]-glucose. Reperfusion-induced loss of NAD(+) and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition-induced NAD(+) loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A. CONCLUSIONS: Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.

Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. METHODS AND RESULTS: In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. CONCLUSIONS: In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

The late phase of preconditioning and its natural clinical application--gene therapy.

There is little doubt that the discovery of ischemic preconditioning (PC) has been one of the fundamental milestones in the field of ischemic biology in the past 20 years. The purpose of this article is to review the pathophysiology and molecular basis of the late phase of myocardial PC. The exploitation of late PC for the development of novel gene therapy strategies aimed at inducing a permanently preconditioned cardiac phenotype (prophylactic cardioprotection) will also be discussed. Deciphering the mechanism of late PC has not only conceptual interest but also a considerable therapeutic implications, since transfer of the genes that underlie late PC would be expected to replicate the salubrious effects of this response of the heart to stress.

The late phase of ischemic preconditioning induces a prosurvival genetic program that results in marked attenuation of apoptosis.

Although the cardioprotection afforded by the late phase of ischemic preconditioning (PC) in ischemia/reperfusion (I/R) injury has been well studied, it is unknown whether this beneficial effect can be attributed to inhibition of apoptosis. We hypothesized that ischemic PC affords protection by suppressing apoptosis and examined the underlying mechanisms. Myocardial infarction was produced in mice (30-min coronary occlusion). In animals preconditioned 24 h earlier with six 4-min coronary occlusion/4-min reperfusion (O/R) cycles, there was a marked decrease in apoptosis as assessed by three different parameters: hairpin-1 assay, caspase-3 activity, and immunohistochemical analysis of active caspase-3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). This protective effect was accompanied by increased expression of multiple antiapoptotic proteins that regulate both the mitochondria-mediated (Bcl-x(L) and Mcl-1) and the death-receptor-mediated (c-FLIP(L) and c-FLIP(S)) pathway of apoptosis and by decreased expression of the proapoptotic protein Bad. This is the first demonstration that the late phase of ischemic PC attenuates cardiac apoptosis after ischemia/reperfusion injury and that this salubrious effect is associated with a complex genetic prosurvival program that results in modulation of several key proteins involved in both the mitochondrial and the death receptor pathways of apoptosis.

Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification.

This study was designed to examine whether NO regulates protein glutathiolation. Exposure to NO donors increased protein glutathiolation in COS-7 or rat aortic smooth muscle cells as detected by anti-protein glutathione (GSH) antibodies. This process was reversible and saturable. Stimulation with acetylcholine (ACh) increased protein glutathiolation in isolated rat aortic rings. This was prevented by inhibiting endothelial NO synthase (eNOS). In ACh-treated rings, proteins showing positive immunoreactivity with the anti-PSSG antibody (Ab) were identified by matrix assisted laser desorption-time-of-flight mass spectrometry to be actin, vimentin, and heat shock protein 70. Purified actin was more readily glutathiolated by S-nitrosoglutathione than by oxidized GSH as determined by electrospray-ionization mass spectrometry, and nitrosylated actin was glutathiolated by reduced GSH. Relative to wild-type (WT) mice, increased protein glutathiolation was observed in hearts of mice with cardiac-specific expression of inducible NO synthase (iNOS). Proteins immunoprecipitated from transgenic hearts revealed GSH-adducted peptides corresponding to adenine nucleotide translocator and the alpha-subunit of F1F0ATPase. These data suggest that exogenous NO or NO generated by eNOS or iNOS regulates protein adduction with GSH. This could be due to a direct reaction of proteins with S-nitrosoglutathione or denitrosylation of S-nitrosylated proteins by reduced GSH. Glutathiolation of cytoskeletal and mitochondrial proteins may be a significant feature of NO bioreactivity.

Prolonged oxidative stress inverts the cardiac force-frequency relation: role of altered calcium handling and myofilament calcium responsiveness.

The normally positive force- and Ca2+ -frequency responses (FFR and CaFR) are inverted in heart failure (HF); whether oxidative stress contributes to these abnormalities is unknown. We evaluated the impact of acute and prolonged oxidative stress on contraction and Ca2+ handling in adult rat cardiomyocytes. Acute (30 min) exposure to H2O2 (100 microM) induced a twofold increase (P<0.025) in intracellular oxyradicals together with contractile depression despite preservation of the Ca2+ transient and the FFR and CaFR to 3 Hz, indicating reduced myofilament Ca2+ responsiveness. In contrast, prolonged (24 h) exposure to the copper-zinc superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC, 1 microM) similarly augmented oxyradicals but also increased cell size, and contraction and Ca2+ transient duration (P<0.025). DDC-treated myocytes displayed inverted FFRs and attenuated (though still positive) CaFRs as compared to control, indicating reduced myofilament Ca2+ responsiveness coupled with altered Ca2+ handling. Protein levels of the Na+ -Ca2+ exchanger (NCX), sarcoplasmic reticular (SR) Ca2+ ATPase (SERCA2), and serine-16 phosphorylated phospholamban (pSer16-PLB) were increased (P<0.025), whereas dihydropyridine receptor abundance was decreased. Total PLB and ryanodine receptor protein expression were unchanged. Caffeine-induced Ca2+ release showed increased NCX activity (P<0.025) without changes in total releasable SR Ca2+, suggesting compensatory changes in SERCA2 and pSer16-PLB to maintain SR Ca2+ load. The superoxide scavenger Tiron attenuated these effects. Thus, acute oxyradical exposure rapidly depresses myofibrillar Ca2+ responsiveness. Prolonged oxidative stress further induces alterations in Ca2+ handling that combined with extant reductions in myofibrillar responsiveness invert the FFR. With regard to Ca2+ handling, reduced transsarcolemmal Ca2+ flux rather than reduced SR Ca2+ uptake was the primary determinant of a negative FFR. Analogous changes may be operative in HF, a state characterized by both oxidative stress and Ca2+ dysregulation.