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Background: Use of sodium-glucose transporter-2 (SGLT2) inhibitors has dramatically increased over the past decade. This medication class predisposes patients to euglycaemic diabetic ketoacidosis, particularly during times of physiologic stress, including fasting and surgery. Beyond case reports and series, a systematic description of perioperative metabolic effects of SGLT2 inhibitors is lacking., Methods: We examined the degree of anion gap acidosis, controlling for non-ketone anions, in patients undergoing surgery at Massachusetts General Hospital in 2016-22. We constructed a multivariable regression model incorporating known non-ketone contributors to the postoperative anion gap (albumin, lactate, estimated glomerular filtration rate, and preoperative anion gap), hold time, and interaction terms between hold time and three previously suggested risk factors for euglycaemic diabetic ketoacidosis: emergency surgery, cardiac surgery, and insulin use., Results: In 463 patients on SGLT2 inhibitors, we observed a strong association between decreased hold time and postoperative anion gap (P<0.001 in a univariable analysis; -0.43, 95% confidence interval [-0.76 to -0.11] change in anion gap per day held, P=0.01 in a multivariable analysis). A significant interaction between hold time and emergency surgery was observed, whereas there was no apparent interaction with insulin use or cardiac surgery., Conclusions: These findings provide the first evidence that an anion gap acidosis, likely from ketoacids, develops in all patients who do not hold SGLT2 inhibitors before surgery rather than in an idiosyncratic few. If an SGLT2 inhibitor is unable to be stopped, postoperative monitoring of anion gap and serum ketones can help detect clinically significant euglycaemic diabetic ketoacidosis, particularly in those undergoing emergency surgery., (Copyright © 2023 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Oxidative stress is associated with cardiovascular and neurodegenerative diseases. Here we report studies of neurovascular oxidative stress in chemogenetic transgenic mouse lines expressing yeast D-amino acid oxidase (DAAO) in neurons and vascular endothelium. When these transgenic mice are fed D-amino acids, DAAO generates hydrogen peroxide in target tissues. DAAO-TG Cdh5 transgenic mice express DAAO under control of the putatively endothelial-specific Cdh5 promoter. When we provide these mice with D-alanine, they rapidly develop sensory ataxia caused by oxidative stress and mitochondrial dysfunction in neurons within dorsal root ganglia and nodose ganglia innervating the heart. DAAO-TG Cdh5 mice also develop cardiac hypertrophy after chronic chemogenetic oxidative stress. This combination of ataxia, mitochondrial dysfunction, and cardiac hypertrophy is similar to findings in patients with Friedreich's ataxia. Our observations indicate that neurovascular oxidative stress is sufficient to cause sensory ataxia and cardiac hypertrophy. Studies of DAAO-TG Cdh5 mice could provide mechanistic insights into Friedreich's ataxia., (© 2023. The Author(s).)

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H 2 O 2 ) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H 2 O 2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart. NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H 2 O 2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.

Chemogenetics refers to experimental systems that dynamically regulate the activity of a recombinant protein by providing or withholding the protein's specific biochemical stimulus. Chemogenetic tools permit precise dynamic control of specific signaling molecules to delineate the roles of those molecules in physiology and disease. Yeast d-amino acid oxidase (DAAO) enables chemogenetic manipulation of intracellular redox balance by generating hydrogen peroxide only in the presence of d-amino acids. Advances in biosensors have allowed the precise quantitation of these signaling molecules. The combination of chemogenetic approaches with biosensor methodologies has opened up new lines of investigation, allowing the analysis of intracellular redox pathways that modulate physiological and pathological cell responses. We anticipate that newly developed transgenic chemogenetic models will permit dynamic modulation of cellularredox balance in diverse cells and tissues and will facilitate the identification and validation of novel therapeutic targets involved in both physiological redox pathways and pathological oxidative stress.

A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H 2 O 2 to living cells or tissues. However, the addition of H 2 O 2 to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of H 2 O 2 on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids. Much has been learned about the intracellular transport of H 2 O 2 through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of H 2 O 2 under hypoxic conditions imaged with the novel ultrasensitive H 2 O 2 sensor (HyPer7). Moreover, this approach was suitable for generating H 2 O 2 in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of d-amino acids differentially affects mDAAO-dependent intracellular H 2 O 2 generation. When paired with the hydrogen sulfide (H 2 S) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust H 2 S signals. We also show that chemogenetic H 2 O 2 generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H 2 O 2 ), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H 2 O 2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H 2 O 2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H 2 O 2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H 2 O 2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Hydrogen peroxide (H 2 O 2 ) modulates critical phosphorylation pathways in vascular endothelial cells, many of which affect endothelial nitric oxide synthase (eNOS) signal transduction. Both intracellular and extracellular sources of H 2 O 2 have been implicated in eNOS regulation, yet the specific endothelial pathways remain incompletely understood. Here we exploited chemogenetic approaches and live-cell imaging methods to both generate and detect H 2 O 2 in different subcellular compartments (cytosol, nucleus, and caveolae) of cultured EA.hy926 human endothelial cells. We developed novel recombinant constructs encoding differentially-targeted yeast d-amino acid oxidase (DAAO), which generates H 2 O 2 only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new H 2 O 2 biosensor HyPer7.2, which allowed us to quantitate intracellular H 2 O 2 levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular H 2 O 2 to the HyPer-DAAO-transfected cells led to increases in H 2 O 2 throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for H 2 O 2 . The sensor response to extracellular H 2 O 2 was more rapid than that quantitated following the addition of d-alanine to transfected cells to activate differentially-targeted DAAO. The maximal intracellular levels of H 2 O 2 observed in response to the addition of extracellular H 2 O 2 vs. intracellular (DAAO-generated) H 2 O 2 were quantitatively similar. Despite these similarities in the measured levels of intracellular H 2 O 2 , we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular H 2 O 2 and the phosphorylation responses elicited by the addition of extracellular H 2 O 2 to the cells. Addition of extracellular H 2 O 2 had only a nominal effect on phosphorylation of eNOS, kinase Akt or AMP-activated protein kinase (AMPK). By contrast, intracellular H 2 O 2 generation by DAAO caused striking increases in the phosphorylation of these same key signaling proteins. We also found that the AMPK inhibitor Compound C completely blocked nuclear H 2 O 2 -promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following H 2 O 2 generation from cytosol- or caveolae-targeted DAAO. We conclude that H 2 O 2 generated in the cell nucleus activates AMPK, leading to eNOS phosphorylation; in contrast, AMPK activation by cytosol- or caveolae-derived H 2 O 2 does not promote eNOS phosphorylation via AMPK. These findings indicate that H 2 O 2 generated in different subcellular compartments differentially modulates endothelial cell phosphorylation pathways, and suggest that dynamic subcellular localization of oxidants may modulate signaling responses in endothelial cells., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Nitric oxide (NO) synthesized by the endothelial isoform of nitric oxide synthase (eNOS) is a critical determinant of vascular homeostasis. However, the real-time detection of intracellular NO-a free radical gas-has been difficult, and surrogate markers for eNOS activation are widely utilized. eNOS phosphorylation can be easily measured in cells by probing immunoblots with phosphospecific antibodies. Here, we pursued multispectral imaging approaches using biosensors to visualize intracellular NO and Ca 2+ and exploited chemogenetic approaches to define the relationships between NO synthesis and eNOS phosphorylation in cultured endothelial cells. We found that the G protein-coupled receptor agonists adenosine triphosphate (ATP) and histamine promoted rapid increases in eNOS phosphorylation, as did the receptor tyrosine kinase agonists insulin and Vascular Endothelial Growth Factor (VEGF). Histamine and ATP also promoted robust NO formation and increased intracellular Ca 2+ By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca 2+ -despite eliciting strong eNOS phosphorylation responses. Our findings demonstrate an unexpected and striking discordance between receptor-modulated eNOS phosphorylation and NO formation in endothelial cells. Previous reports in which phosphorylation of eNOS has been studied as a surrogate for enzyme activation may need to be reassessed., Competing Interests: The authors declare no conflict of interest.

Cellular antioxidant systems control the levels of hydrogen peroxide (H 2 O 2 ) within cells. Multiple theoretical models exist that predict the diffusion properties of H 2 O 2 depending on the rate of H 2 O 2 generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H 2 O 2 gradients. The relative role of thioredoxin (Trx) and glutathione systems in H 2 O 2 pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H 2 O 2 gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H 2 O 2 and using the H 2 O 2 biosensor HyPer to analyze the resulting H 2 O 2 distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H 2 O 2 gradients. Antioxid. Redox Signal. 31, 664-670.

We previously described a novel "chemogenetic" animal model of heart failure that recapitulates a characteristic feature commonly found in human heart failure: chronic oxidative stress. This heart failure model uses a chemogenetic approach to activate a recombinant yeast d-amino acid oxidase in rat hearts in vivo to generate oxidative stress, which then rapidly leads to the development of a dilated cardiomyopathy. Here we apply this new model to drug testing by studying its response to treatment with the angiotensin II (ANG II) receptor blocker valsartan, administered either alone or with the neprilysin inhibitor sacubitril. Echocardiographic and [ 18 F]fluorodeoxyglucose positron emission tomographic imaging revealed that valsartan in the presence or absence of sacubitril reverses the anatomical and metabolic remodeling induced by chronic oxidative stress. Markers of oxidative stress, mitochondrial function, and apoptosis, as well as classical heart failure biomarkers, also normalized following drug treatments despite the persistence of cardiac fibrosis. These findings provide evidence that chemogenetic heart failure is rapidly reversible by drug treatment, setting the stage for the study of novel heart failure therapeutics in this model. The ability of ANG II blockade and neprilysin inhibition to reverse heart failure induced by chronic oxidative stress identifies a central role for cardiac myocyte angiotensin receptors in the pathobiology of cardiac dysfunction caused by oxidative stress. NEW & NOTEWORTHY The chemogenetic approach allows us to distinguish cardiac myocyte-specific pathology from the pleiotropic changes that are characteristic of other "interventional" animal models of heart failure. These features of the chemogenetic heart failure model facilitate the analysis of drug effects on the progression and regression of ventricular remodeling, fibrosis, and dysfunctional signal transduction. Chemogenetic approaches will be highly informative in the study of the roles of redox stress in heart failure providing an opportunity for the identification of novel therapeutic targets.

Oxidative stress plays an important role in the pathogenesis of many disease states. In the heart, reactive oxygen species are linked with cardiac ischemia/reperfusion injury, hypertrophy, and heart failure. While this correlation between ROS and cardiac pathology has been observed in multiple models of heart failure, the independent role of hydrogen peroxide (H 2 O 2 ) in vitro and in vivo is unclear, owing to a lack of tools for precise manipulation of intracellular redox state. Here we apply a chemogenetic system based on a yeast D-amino acid oxidase to show that chronic generation of H 2 O 2 in the heart induces a dilated cardiomyopathy with significant systolic dysfunction. We anticipate that chemogenetic approaches will enable future studies of in vivo H 2 O 2 signaling not only in the heart, but also in the many other organ systems where the relationship between redox events and physiology remains unclear.

Rationale: Hydrogen peroxide (H 2 O 2 ) is a stable reactive oxygen species (ROS) that has long been implicated in insulin signal transduction in adipocytes. However, H 2 O 2 's role in mediating insulin's effects on the heart are unknown., Objective: We investigated the role of H 2 O 2 in activating insulin-dependent changes in cardiac myocyte metabolic and inotropic pathways. The sources of insulin-dependent H 2 O 2 generation were also studied., Methods and Results: In addition to the canonical role of insulin in modulating cardiac metabolic pathways, we found that insulin also inhibited beta adrenergic-induced increases in cardiac contractility. Catalase and NADPH oxidase (NOX) inhibitors blunted activation of insulin-responsive kinases Akt and mTOR and attenuated beta adrenergic receptor-mediated responses. These insulin responses were lost in a mouse model of type 2 diabetes, suggesting a role for these H 2 O 2 -dependent pathways in the diabetic heart. The H 2 O 2 -sensitive fluorescent biosensor HyPer revealed rapid increases in cytosolic and caveolar H 2 O 2 concentrations in response to insulin treatment, which were blocked by NOX inhibitors and attenuated in NOX2 KO and NOX4 KO mice. In NOX2 KO cardiac myocytes, insulin-mediated phosphorylation of Akt and mTOR was blocked, while these responses were unaffected in cardiac myocytes from NOX4 KO mice. In contrast, insulin's effects on contractility were lost in cardiac myocytes from NOX4 KO animals but were retained in NOX2 KO mice., Conclusions: These studies identify a proximal point of bifurcation in cardiac insulin signaling through the simultaneous activation of both NOX2 and NOX4. Each NOX isoform generates H 2 O 2 in cardiac myocytes with distinct time courses, with H 2 O 2 derived from NOX2 augmenting Akt-dependent metabolic effects of insulin, while H 2 O 2 from NOX4 blocks beta adrenergic increases in inotropy. These findings suggest that insulin resistance in the diabetic heart may lead to potentially deleterious potentiation of beta adrenergic responses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Cooling following cardiac arrest can improve survival significantly. However, delays in achieving target temperature may decrease the overall benefits of cooling. Here, we test whether lipid emulsion, a clinically approved drug reported to exert cardioprotection, can rescue heart contractility in the setting of delayed cooling in stunned mouse cardiomyocytes., Design: Cell culture study., Setting: Academic research laboratory., Subjects: Cardiomyocytes isolated from 1- to 2-day-old C57BL6 mice., Interventions: Cardiomyocytes were exposed to 30 minutes of ischemia followed by 90 minutes of reperfusion and 10 minutes of isoproterenol with nine interventions: 1) no additional treatment; 2) intraischemic cooling at 32 °C initiated 10 minutes prior to reperfusion; 3) delayed cooling started 20 minutes after reperfusion; 4) lipid emulsion + delayed cooling; 5) lipid emulsion (0.25%) administered at reperfusion; 6) lipid emulsion + intraischemic cooling; 7) delayed lipid emulsion; 8) lipid emulsion + delayed cooling + Akt inhibitor (API-2, 10 µM); and 9) lipid emulsion + delayed cooling + Erk inhibitor (U0126, 10 µM). Inhibitors were given to cells 1 hour prior to ischemia., Measurements and Main Results: Contractility was recorded by real-time phase-contrast imaging and analyzed with pulse image velocimetry in MATLAB (Mathworks, Natick, MA). Ischemia diminished cell contraction. The cardioprotective effect of cooling was diminished when delayed but was rescued by lipid emulsion. Further, lipid emulsion on its own improved recovery of the contractility to a greater extent as intraischemic cooling. However, cotreatment of lipid emulsion and intraischemic cooling did not further improve the recovery compared to either treatment alone. Furthermore, Akt and Erk inhibitors blocked lipid emulsion-induced protection., Conclusions: Lipid emulsion improved contractility and rescued contractility in the context of delayed cooling. This protective effect required Akt and Erk signaling. Lipid emulsion might serve as a treatment or adjunct to cooling in ameliorating myocardial ischemia/reperfusion injury.