Alison C. Brewer

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

Endothelial Nox4 NADPH Oxidase Enhances Vasodilatation and Reduces Blood Pressure In Vivo

Objective—Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H2O2), but its role in vivo remains unclear. Methods and Results—We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H2O2 production and H2O2-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. Conclusion—Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H2O2 production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

Redox signaling in cardiac myocytes

The heart has complex mechanisms that facilitate the maintenance of an oxygen supply–demand balance necessary for its contractile function in response to physiological fluctuations in workload as well as in response to chronic stresses such as hypoxia, ischemia, and overload. Redox-sensitive signaling pathways are centrally involved in many of these homeostatic and stress-response mechanisms. Here, we review the main redox-regulated pathways that are involved in cardiac myocyte excitation–contraction coupling, differentiation, hypertrophy, and stress responses. We discuss specific sources of endogenously generated reactive oxygen species (e.g., mitochondria and NADPH oxidases of the Nox family), the particular pathways and processes that they affect, the role of modulators such as thioredoxin, and the specific molecular mechanisms that are involved—where this knowledge is available. A better understanding of this complex regulatory system may allow the development of more specific therapeutic strategies for heart diseases.

Nox4 and Nox2 NADPH Oxidases Mediate Distinct Cellular Redox Signaling Responses to Agonist Stimulation

Objectives—The NADPH oxidase isoforms Nox2 and Nox4 are coexpressed in many cell types and are implicated in agonist-stimulated redox-sensitive signal transduction. We compared the involvement of Nox2 versus Nox4 in redox-sensitive protein kinase activation after agonist stimulation. Methods and Results—We transfected HEK293 cells with Nox2 or Nox4 and compared ROS production and activation of mitogen activated protein kinases (MAPKs), Akt, and GSK3β after acute agonist stimulation. Nox4 overexpression substantially increased basal ROS generation whereas ROS generation in response to angiotensin II and tumor necrosis factor (TNF)α was enhanced in Nox2-overexpressing cells. Nox4 overexpression induced basal activation of ERK1/2 and JNK whereas Nox2-transfected cells showed a modest increase in p38MAPK activation. After angiotensin II or TNFα treatment, JNK activation was augmented in Nox2 but not Nox4-transfected cells, whereas insulin augmented phosphorylation of p38MAPK, Akt, and GSK3β specifically in Nox4-overexpressing cells and JNK specifically in Nox2-overexpressing cells. Conclusions—These data indicate that Nox2 and Nox4 exhibit distinctive patterns of acute activation by angiotensin II, TNFα, and insulin and regulate the activation of distinct protein kinases.

GATA factors in vertebrate heart development and disease

Vertebrate heart formation is dependent upon complex hierarchical gene regulatory networks, which effect both the specification and differentiation of cardiomyocytes and subsequently cardiac morphogenesis. GATA-4, -5 and -6 comprise an evolutionarily conserved subfamily of transcription factors, which are expressed within the precardiac mesoderm from early stages in its specification and continue to be expressed within the adult heart. We review here the functional roles of individual GATA transcription factors in cardiac development, normal homeostasis and disease. We also review the cellular mechanisms employed to regulate the expression and downstream targets of the different GATA factors.

NADPH oxidase signaling and cardiac myocyte function

The NADPH oxidase family of enzymes has emerged as a major source of reactive oxygen species (ROS) that is important in diverse cellular functions including anti-microbial defence, inflammation and redox signaling. Of the five known NADPH oxidase isoforms, several are expressed in cardiovascular cells where they are involved in physiological and pathological processes such as the regulation of vascular tone, cell growth, migration, proliferation, hypertrophy, apoptosis and matrix deposition. This article reviews current knowledge regarding the role of NADPH oxidases in cardiomyocyte function in health and disease.

Activation of TRPC6 Channels Is Essential for Lung Ischaemia–Reperfusion Induced Oedema in Mice

Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension.

Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo

NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.

The human and mouse GATA-6 genes utilize two promoters and two initiation codons

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5′ noncoding exons are utilized. Both transcript isoforms are expressed in the same tissue-specific and developmental stage-specific pattern, and their ratio appears similar wherever examined. The more upstream noncoding exon showed a substantial degree of homology between the two mammalian species, suggesting a conserved regulatory function. Moreover, in transfection assays we show that elements within this exon act to promote its transcription. Positive regulatory elements that effect transcription from the more downstream exon were not apparent in this assay, revealing a regulatory distinction between the two promoters. We also demonstrate alternative initiator codon usage in both the human and mouse GATA-6 genes. Both isoforms of the protein are synthesized in vitro regardless of which 5′ noncoding exon is present in the RNA, although the larger protein has greater transcriptional activation potential in transfection assays. Thus, GATA-6 function in the cell is controlled by a complex interplay of transcriptional and translational regulation.