Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

Rethinking False Positive Exercise Electrocardiographic Stress Tests by Assessing Coronary Microvascular Function.

BACKGROUND: Exercise electrocardiographic stress testing (EST) has historically been validated against the demonstration of obstructive coronary artery disease. However, myocardial ischemia can occur because of coronary microvascular dysfunction (CMD) in the absence of obstructive coronary artery disease. OBJECTIVES: The aim of this study was to assess the specificity of EST to detect an ischemic substrate against the reference standard of coronary endothelium-independent and endothelium-dependent microvascular function in patients with angina with nonobstructive coronary arteries (ANOCA). METHODS: Patients with ANOCA underwent invasive coronary physiological assessment using adenosine and acetylcholine. CMD was defined as impaired endothelium-independent and/or endothelium-dependent function. EST was performed using a standard Bruce treadmill protocol, with ischemia defined as the appearance of ≥0.1-mV ST-segment depression 80 ms from the J-point on electrocardiography. The study was powered to detect specificity of ≥91%. RESULTS: A total of 102 patients were enrolled (65% women, mean age 60 ± 8 years). Thirty-two patients developed ischemia (ischemic group) during EST, whereas 70 patients did not (nonischemic group); both groups were phenotypically similar. Ischemia during EST was 100% specific for CMD. Acetylcholine flow reserve was the strongest predictor of ischemia during exercise. Using endothelium-independent and endothelium-dependent microvascular dysfunction as the reference standard, the false positive rate of EST dropped to 0%. CONCLUSIONS: In patients with ANOCA, ischemia on EST was highly specific of an underlying ischemic substrate. These findings challenge the traditional belief that EST has a high false positive rate.

Characterizing Mechanisms of Ischemia in Patients With Myocardial Bridges.

BACKGROUND: Myocardial bridges (MBs) are prevalent and can be associated with acute and chronic ischemic syndromes. We sought to determine the substrates for ischemia in patients with angina with nonobstructive coronary arteries and a MB in the left anterior descending artery. METHODS: Patients with angina with nonobstructive coronary arteries underwent the acquisition of intracoronary pressure and flow during rest, supine bicycle exercise, and adenosine infusion. Coronary wave intensity analysis was performed, with perfusion efficiency defined as accelerating wave energy/total wave energy (%). Epicardial endothelial dysfunction was defined as a reduction in epicardial vessel diameter ≥20% in response to intracoronary acetylcholine infusion. Patients with angina with nonobstructive coronary arteries and a MB were compared with 2 angina with nonobstructive coronary arteries groups with no MB: 1 with coronary microvascular disease (CMD: coronary flow reserve, <2.5) and 1 with normal coronary flow reserve (reference: coronary flow reserve, ≥2.5). RESULTS: Ninety-two patients were enrolled in the study (30 MB, 33 CMD, and 29 reference). Fractional flow reserve in these 3 groups was 0.86±0.05, 0.92±0.04, and 0.94±0.05; coronary flow reserve was 2.5±0.5, 2.0±0.3, and 3.2±0.6. Perfusion efficiency increased numerically during exercise in the reference group (65±9%-69±13%; P=0.063) but decreased in the CMD (68±10%-50±10%; P<0.001) and MB (66±9%-55±9%; P<0.001) groups. The reduction in perfusion efficiency had distinct causes: in CMD, this was driven by microcirculation-derived energy in early diastole, whereas in MB, this was driven by diminished accelerating wave energy, due to the upstream bridge, in early systole. Epicardial endothelial dysfunction was more common in the MB group (54% versus 29% reference and 38% CMD). Overall, 93% of patients with a MB had an identifiable ischemic substrate. CONCLUSIONS: MBs led to impaired coronary perfusion efficiency during exercise, which was due to diminished accelerating wave energy in early systole compared with the reference group. Additionally, there was a high prevalence of endothelial and microvascular dysfunction. These ischemic mechanisms may represent distinct treatment targets.

Diagnostic accuracy, clinical characteristics, and prognostic differences of patients with acute myocarditis according to inclusion criteria.

INTRODUCTION: The diagnosis of acute myocarditis (AM) is complex due to its heterogeneity and typically is defined by either Electronic Healthcare Records (EHRs) or advanced imaging and endomyocardial biopsy, but there is no consensus. We aimed to investigate the diagnostic accuracy of these approaches for AM. METHODS: Data on ICD 10th Revision(ICD-10) codes corresponding to AM were collected from two hospitals and compared to CMR-confirmed or clinically suspected(CS) AM cases with respect to diagnostic accuracy, clinical characteristics, and all-cause mortality. Next, we performed a review of published AM studies according to inclusion criteria. RESULTS: We identified 291 unique admissions with ICD-10 codes corresponding to AM in the first three diagnostic positions. The positive predictive value(PPV) of ICD-10 codes for CMR-confirmed or CS-AM was 36%, and patients with CMR-confirmed or CS AM had a lower all-cause mortality than those with a refuted diagnosis (P = 0.019). Using an unstructured approach, patients with CMR-confirmed and CS AM had similar demographics, comorbidity profiles and survival over a median follow-up of 52 months (P = 0.72). Our review of the literature confirmed our findings. Outcomes for patients included in studies using CMR-confirmed criteria were favourable compared to studies with EMB-confirmed AM cases. CONCLUSION: ICD-10 codes have poor accuracy in identification of AM cases and should be used with caution in clinical research. There are important differences in management and outcomes of patients according to the selection criteria used to diagnose AM. Potential selection biases must be considered when interpreting AM cohorts and requires standardisation of inclusion criteria for AM studies.

Health position paper and redox perspectives on reactive oxygen species as signals and targets of cardioprotection.

The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca2+ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal.

Nicotinamide Adenosine Dinucleotide Phosphate Oxidase-Mediated Signaling in Cardiac Remodeling.

Significance: Reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling and the subsequent progression to heart failure (HF). Nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases (NOXs) are one of the major sources of ROS and are expressed in different heart cell types, including cardiomyocytes, endothelial cells, fibroblasts, and inflammatory cells. Recent Advances: NOX-derived ROS are usually produced in a regulated and spatially confined fashion and typically linked to specific signaling. The two main cardiac isoforms, namely nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) and nicotinamide adenine dinucleotide phosphate oxidase isoform 4 (NOX4), possess different biochemical and (patho)physiological properties and exert distinct effects on the cardiac phenotype in many settings. Recent work has defined important cell-specific effects of NOX2 that contribute to pathological cardiac remodeling and dysfunction. NOX4, on the other hand, may exert protective effects by stimulating adaptive stress responses, with recent data showing that NOX4-mediated signaling regulates transcription and metabolism in the heart. Critical Issues: The inhibition of NOX2 appears to be a very promising therapeutic target to ameliorate pathological cardiac remodeling. If the beneficial effects of NOX4 can be enhanced, this might be a unique approach to boosting adaptive responses and thereby impact cell survival, activation, contractility, and growth. Future Directions: Increasing knowledge regarding the intricacies of NOX-mediated signaling may yield tractable therapeutic targets, in contrast to the non-specific targeting of oxidative stress. Antioxid. Redox Signal. 38, 371-387.

Coronary Wave Intensity Analysis as an Invasive and Vessel-Specific Index of Myocardial Viability.

BACKGROUND: Coronary angiography and viability testing are the cornerstones of diagnosing and managing ischemic cardiomyopathy. At present, no single test serves both needs. Coronary wave intensity analysis interrogates both contractility and microvascular physiology of the subtended myocardium and therefore has the potential to fulfil the goal of completely assessing coronary physiology and myocardial viability in a single procedure. We hypothesized that coronary wave intensity analysis measured during coronary angiography would predict viability with a similar accuracy to late-gadolinium-enhanced cardiac magnetic resonance imaging. METHODS: Patients with a left ventricular ejection fraction ≤40% and extensive coronary disease were enrolled. Coronary wave intensity analysis was assessed during cardiac catheterization at rest, during adenosine-induced hyperemia, and during low-dose dobutamine stress using a dual pressure-Doppler sensing coronary guidewire. Scar burden was assessed with cardiac magnetic resonance imaging. Regional left ventricular function was assessed at baseline and 6-month follow-up after optimization of medical-therapy±revascularization, using transthoracic echocardiography. The primary outcome was myocardial viability, determined by the retrospective observation of functional recovery. RESULTS: Forty participants underwent baseline physiology, cardiac magnetic resonance imaging, and echocardiography, and 30 had echocardiography at 6 months; 21/42 territories were viable on follow-up echocardiography. Resting backward compression wave energy was significantly greater in viable than in nonviable territories (-5240±3772 versus -1873±1605 W m-2 s-1, P<0.001), and had comparable accuracy to cardiac magnetic resonance imaging for predicting viability (area under the curve 0.812 versus 0.757, P=0.649); a threshold of -2500 W m-2 s-1 had 86% sensitivity and 76% specificity. CONCLUSIONS: Backward compression wave energy has accuracy similar to that of late-gadolinium-enhanced cardiac magnetic resonance imaging in the prediction of viability. Coronary wave intensity analysis has the potential to streamline the management of ischemic cardiomyopathy, in a manner analogous to the effect of fractional flow reserve on the management of stable angina.

Cardiometabolic outcomes up to 12 months after COVID-19 infection. A matched cohort study in the UK.

BACKGROUND: Acute Coronavirus Disease 2019 (COVID-19) has been associated with new-onset cardiovascular disease (CVD) and diabetes mellitus (DM), but it is not known whether COVID-19 has long-term impacts on cardiometabolic outcomes. This study aimed to determine whether the incidence of new DM and CVDs are increased over 12 months after COVID-19 compared with matched controls. METHODS AND FINDINGS: We conducted a cohort study from 2020 to 2021 analysing electronic records for 1,356 United Kingdom family practices with a population of 13.4 million. Participants were 428,650 COVID-19 patients without DM or CVD who were individually matched with 428,650 control patients on age, sex, and family practice and followed up to January 2022. Outcomes were incidence of DM and CVD. A difference-in-difference analysis estimated the net effect of COVID-19 allowing for baseline differences, age, ethnicity, smoking, body mass index (BMI), systolic blood pressure, Charlson score, index month, and matched set. Follow-up time was divided into 4 weeks from index date ("acute COVID-19"), 5 to 12 weeks from index date ("post-acute COVID-19"), and 13 to 52 weeks from index date ("long COVID-19"). Net incidence of DM increased in the first 4 weeks after COVID-19 (adjusted rate ratio, RR 1.81, 95% confidence interval (CI) 1.51 to 2.19) and remained elevated from 5 to 12 weeks (RR 1.27, 1.11 to 1.46) but not from 13 to 52 weeks overall (1.07, 0.99 to 1.16). Acute COVID-19 was associated with net increased CVD incidence (5.82, 4.82 to 7.03) including pulmonary embolism (RR 11.51, 7.07 to 18.73), atrial arrythmias (6.44, 4.17 to 9.96), and venous thromboses (5.43, 3.27 to 9.01). CVD incidence declined from 5 to 12 weeks (RR 1.49, 1.28 to 1.73) and showed a net decrease from 13 to 52 weeks (0.80, 0.73 to 0.88). The analyses were based on health records data and participants' exposure and outcome status might have been misclassified. CONCLUSIONS: In this study, we found that CVD was increased early after COVID-19 mainly from pulmonary embolism, atrial arrhythmias, and venous thromboses. DM incidence remained elevated for at least 12 weeks following COVID-19 before declining. People without preexisting CVD or DM who suffer from COVID-19 do not appear to have a long-term increase in incidence of these conditions.

Implications of elevated troponin on time-to-surgery in non-ST elevation myocardial infarction (NIHR Health Informatics Collaborative: TROP-CABG study).

Implications of elevated troponin on time-to-surgery in non-ST elevation myocardial infarction(NIHR Health Informatics Collaborative:TROP-CABG study). Benedetto et al. BACKGROUND: The optimal timing of coronary artery bypass grafting (CABG) in patients with non-ST elevation myocardial infarction (NSTEMI) and the utility of pre-operative troponin levels in decision-making remains unclear. We investigated (a) the association between peak pre-operative troponin and survival post-CABG in a large cohort of NSTEMI patients and (b) the interaction between troponin and time-to-surgery. METHODS AND RESULTS: Our cohort consisted of 1746 patients (1684 NSTEMI; 62 unstable angina) (mean age 69 ± 11 years,21% female) with recorded troponins that had CABG at five United Kingdom centers between 2010 and 2017. Time-segmented Cox regression was used to investigate the interaction of peak troponin and time-to-surgery on early (within 30 days) and late (beyond 30 days) survival. Average interval from peak troponin to surgery was 9 ± 15 days, with 1466 (84.0%) patients having CABG during the same admission. Sixty patients died within 30-days and another 211 died after a mean follow-up of 4 ± 2 years (30-day survival 0.97 ± 0.004 and 5-year survival 0.83 ± 0.01). Peak troponin was a strong predictor of early survival (adjusted P = 0.002) with a significant interaction with time-to-surgery (P interaction = 0.007). For peak troponin levels <100 times the upper limit of normal, there was no improvement in early survival with longer time-to-surgery. However, in patients with higher troponins, early survival increased progressively with a longer time-to-surgery, till day 10. Peak troponin did not influence survival beyond 30 days (adjusted P = 0.64). CONCLUSIONS: Peak troponin in NSTEMI patients undergoing CABG was a significant predictor of early mortality, strongly influenced the time-to-surgery and may prove to be a clinically useful biomarker in the management of these patients.

Doxorubicin induces cardiotoxicity in a pluripotent stem cell model of aggressive B cell lymphoma cancer patients.

Cancer therapies with anthracyclines have been shown to induce cardiovascular complications. The aims of this study were to establish an in vitro induced pluripotent stem cell model (iPSC) of anthracycline-induced cardiotoxicity (ACT) from patients with an aggressive form of B-cell lymphoma and to examine whether doxorubicin (DOX)-treated ACT-iPSC cardiomyocytes (CM) can recapitulate the clinical features exhibited by patients, and thus help uncover a DOX-dependent pathomechanism. ACT-iPSC CM generated from individuals with CD20+ B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction were studied and compared to control-iPSC CM from cancer survivors without cardiac symptoms. In cellular studies, ACT-iPSC CM were persistently more susceptible to DOX toxicity including augmented disorganized myofilament structure, changed mitochondrial shape, and increased apoptotic events. Consistently, ACT-iPSC CM and cardiac fibroblasts isolated from fibrotic human ACT myocardium exhibited higher DOX-dependent reactive oxygen species. In functional studies, Ca2+ transient amplitude of ACT-iPSC CM was reduced compared to control cells, and diastolic sarcoplasmic reticulum Ca2+ leak was DOX-dependently increased. This could be explained by overactive CaMKIIδ in ACT CM. Together with DOX-dependent augmented proarrhythmic cellular triggers and prolonged action potentials in ACT CM, this suggests a cellular link to arrhythmogenic events and contractile dysfunction especially found in ACT engineered human myocardium. CamKIIδ inhibition prevented proarrhythmic triggers in ACT. In contrast, control CM upregulated SERCA2a expression in a DOX-dependent manner, possibly to avoid heart failure conditions. In conclusion, we developed the first human patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma. Our results suggest that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and finally in heart failure after persistent stress activation in ACT patients.

Nox2 underpins microvascular inflammation and vascular contributions to cognitive decline.

Chronic microvascular inflammation and oxidative stress are inter-related mechanisms underpinning white matter disease and vascular cognitive impairment (VCI). A proposed mediator is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2), a major source of reactive oxygen species (ROS) in the brain. To assess the role of Nox2 in VCI, we studied a tractable model with white matter pathology and cognitive impairment induced by bilateral carotid artery stenosis (BCAS). Mice with genetic deletion of Nox2 (Nox2 KO) were compared to wild-type (WT) following BCAS. Sustained BCAS over 12 weeks in WT mice induced Nox2 expression, indices of microvascular inflammation and oxidative damage, along with white matter pathology culminating in a marked cognitive impairment, which were all protected by Nox2 genetic deletion. Neurovascular coupling was impaired in WT mice post-BCAS and restored in Nox2 KO mice. Increased vascular expression of chemoattractant mediators, cell-adhesion molecules and endothelial activation factors in WT mice post-BCAS were ameliorated by Nox2 deficiency. The clinical relevance was confirmed by increased vascular Nox2 and indices of microvascular inflammation in human post-mortem subjects with cerebral vascular disease. Our results support Nox2 activity as a critical determinant of VCI, whose targeting may be of therapeutic benefit in cerebral vascular disease.

Cardiac energetics in patients with chronic heart failure and iron deficiency: an in-vivo 31 P magnetic resonance spectroscopy study.

AIMS: Iron deficiency (ID) is prevalent and adverse in chronic heart failure (CHF) but few human studies have explored the myocardial mechanism(s) that potentially underlie this adversity. Because mitochondrial oxidative phosphorylation (OXPHOS) provides over 90% of the hearts adenosine triphosphate (ATP), and iron is critical for OXPHOS, we hypothesized that patients with CHF and ID would harbour greater cardiac energetic impairments than patients without ID. METHODS AND RESULTS: Phosphorus magnetic resonance spectroscopy was used to quantify the phosphocreatine (PCr) to ATP (PCr/ATP) ratio, an index of in-vivo cardiac energetics, in CHF patients and healthy volunteers. Cardiac structure and function was assessed from magnetic resonance short stack cines. Patients with (n = 27) and without (n = 12) ID, and healthy volunteers (n = 11), were similar with respect to age and gender. The PCr/ATP ratio was lower in patients with ID (1.03 [0.83-1.38]) compared to those without ID (1.72 [1.51-2.26], p < 0.01) and healthy volunteers (1.39 [1.10-3.68], p < 0.05). This was despite no difference in cardiac structure and function between patients with and without ID, and despite adjustment for the presence of anaemia, haemoglobin levels, cardiac rhythm, or New York Heart Association (NYHA) class. In the total CHF cohort, the PCr/ATP ratio correlated with ferritin levels (rho = 0.4, p < 0.01), and was higher in NYHA class I than class II or III patients (p = 0.02). CONCLUSION: Iron deficiency is associated with greater cardiac energetic impairment in patients with CHF irrespective of anaemia and cardiac structure and function. Suppression of cardiac mitochondrial function might therefore be a mechanism via which ID worsens human CHF.

Role of oxidative stress in calcific aortic valve disease and its therapeutic implications.

Calcific aortic valve disease (CAVD) is the end result of active cellular processes that lead to the progressive fibrosis and calcification of aortic valve leaflets. In western populations, CAVD is a significant cause of cardiovascular morbidity and mortality, and in the absence of effective drugs, it will likely represent an increasing disease burden as populations age. As there are currently no pharmacological therapies available for preventing, treating, or slowing the development of CAVD, understanding the mechanisms underlying the initiation and progression of the disease is important for identifying novel therapeutic targets. Recent evidence has emerged of an important causative role for reactive oxygen species (ROS)-mediated oxidative stress in the pathophysiology of CAVD, inducing the differentiation of valve interstitial cells into myofibroblasts and then osteoblasts. In this review, we focus on the roles and sources of ROS driving CAVD and consider their potential as novel therapeutic targets for this debilitating condition.

Systemic inflammation and oxidative stress contribute to acute kidney injury after transcatheter aortic valve implantation.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication of transcatheter aortic valve implantation (TAVI) and has been linked to preexisting comorbidities, peri-procedural hypotension, and systemic inflammation. The extent of systemic inflammation after TAVI is not fully understood. Our aim was to characterize the inflammatory response after TAVI and evaluate its contribution to the mechanism of post-procedural AKI. METHODS: One hundred and five consecutive patients undergoing TAVI at our institution were included. We analyzed the peri-procedural inflammatory and oxidative stress responses by measuring a range of biomarkers (including C-reactive protein [hsCRP], cytokine levels, and myeloperoxidase [MPO]), before TAVI and 6, 24, and 48 hours post-procedure. We correlated this with changes in renal function and patient and procedural characteristics. RESULTS: We observed a significant increase in plasma levels of pro-inflammatory cytokines (hsCRP, interleukin 6, tumor necrosis factor alpha receptors) and markers of oxidative stress (MPO) after TAVI. The inflammatory response was significantly greater after transapical (TA) TAVI compared to transfemoral (TF). This was associated with a higher incidence of AKI in the TA cohort compared to TF (44% vs. 8%, respectively, p < 0.0001). The incidence of AKI was significantly lower when N-acetylcysteine (NAC) was given peri-procedurally (12% vs. 38%, p < 0.005). In multivariate analysis, only the TA approach and no use of NAC before the procedure were independent predictors of AKI. CONCLUSIONS: TAVI creates a significant post-procedural inflammatory response, more so with the TA approach. Mechanisms of AKI after TAVI are complex. Inflammatory response, hypoperfusion, and oxidative stress may all play a part and are potential therapeutic targets to reduce/prevent AKI.

Enhanced Heart Failure in Redox-Dead Cys17Ser PKARIα Knock-In Mice.

Background PKARIα (protein kinase A type I-α regulatory subunit) is redox-active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation-contraction coupling. Methods and Results We used a redox-dead transgenic mouse model with homozygous knock-in replacement of redox-sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long-term relevance of oxidized PKARIα was investigated in KI mice and their wild-type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A-dependent stimulation of ICa (L-type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction-related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of ICa stimulation. Conversely, direct pharmacological stimulation of ICa using Bay K8644 rescued Ca transients in AngII-treated KI myocytes and contractile function in failing KI mice in vivo. Conclusions Oxidative activation of PKARIα with subsequent stimulation of ICa preserves cardiac function in the setting of acute and chronic oxidative stress.

Nox2-deficient Tregs improve heart transplant outcomes via their increased graft recruitment and enhanced potency.

Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodeling. As Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesized that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. We generated a potentially novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+ mice) and transplanted with hearts from CB6F1 donors. As compared with those of littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates, and diminished cardiomyocyte necrosis and allograft fibrosis. Single-cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared with Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR-214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.

Endothelial Nox2 Limits Systemic Inflammation and Hypotension in Endotoxemia by Controlling Expression of Toll-Like Receptor 4.

ABSTRACT: Leukocyte Nox2 is recognized to have a fundamental microbicidal function in sepsis but the specific role of Nox2 in endothelial cells (EC) remains poorly elucidated. Here, we tested the hypothesis that endothelial Nox2 participates in the pathogenesis of systemic inflammation and hypotension induced by LPS. LPS was injected intravenously in mice with Tie2-targeted deficiency or transgenic overexpression of Nox2. Mice with Tie2-targeted Nox2 deficiency had increased circulating levels of TNF-α, enhanced numbers of neutrophils trapped in lungs, and aggravated hypotension after LPS injection, as compared to control LPS-injected animals. In contrast, Tie2-driven Nox2 overexpression attenuated inflammation and prevented the hypotension induced by LPS. Because Tie2-Cre targets both EC and myeloid cells we generated bone marrow chimeric mice with Nox2 deletion restricted to leukocytes or ECs. Mice deficient in Nox2 either in leukocytes or ECs had reduced LPS-induced neutrophil trapping in the lungs and lower plasma TNF-α levels as compared to control LPS-injected mice. However, the pronounced hypotensive response to LPS was present only in mice with EC-specific Nox2 deletion. Experiments in vitro with human vein or aortic endothelial cells (HUVEC and HAEC, respectively) treated with LPS revealed that EC Nox2 controls NF-κB activation and the transcription of toll-like receptor 4 (TLR4), which is the recognition receptor for LPS. In conclusion, these results suggest that endothelial Nox2 limits NF-κB activation and TLR4 expression, which in turn attenuates the severity of hypotension and systemic inflammation induced by LPS.

SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care.

Prognostic characteristics inform risk stratification in intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19). We obtained blood samples (n = 474) from hospitalized COVID-19 patients (n = 123), non-COVID-19 ICU sepsis patients (n = 25) and healthy controls (n = 30). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in plasma or serum (RNAemia) of COVID-19 ICU patients when neutralizing antibody response was low. RNAemia is associated with higher 28-day ICU mortality (hazard ratio [HR], 1.84 [95% CI, 1.22-2.77] adjusted for age and sex). RNAemia is comparable in performance to the best protein predictors. Mannose binding lectin 2 and pentraxin-3 (PTX3), two activators of the complement pathway of the innate immune system, are positively associated with mortality. Machine learning identified 'Age, RNAemia' and 'Age, PTX3' as the best binary signatures associated with 28-day ICU mortality. In longitudinal comparisons, COVID-19 ICU patients have a distinct proteomic trajectory associated with mortality, with recovery of many liver-derived proteins indicating survival. Finally, proteins of the complement system and galectin-3-binding protein (LGALS3BP) are identified as interaction partners of SARS-CoV-2 spike glycoprotein. LGALS3BP overexpression inhibits spike-pseudoparticle uptake and spike-induced cell-cell fusion in vitro.

Rotigaptide Infusion for the First 7 Days After Myocardial Infarction-Reperfusion Reduced Late Complexity of Myocardial Architecture of the Healing Border-Zone and Arrhythmia Inducibility.

Background Survivors of myocardial infarction are at increased risk of late ventricular arrhythmias, with infarct size and scar heterogeneity being key determinants of arrhythmic risk. Gap junctions facilitate the passage of small ions and morphogenic cell signaling between myocytes. We hypothesized that gap junctions enhancement during infarction-reperfusion modulates structural and electrophysiological remodeling and reduces late arrhythmogenesis. Methods and Results Infarction-reperfusion surgery was carried out in male Sprague-Dawley rats followed by 7 days of rotigaptide or saline administration. The in vivo and ex vivo arrhythmogenicity was characterized by programmed electrical stimulation 3 weeks later, followed by diffusion-weighted magnetic resonance imaging and Masson's trichrome histology. Three weeks after 7-day postinfarction administration of rotigaptide, ventricular tachycardia/ventricular fibrillation was induced on programmed electrical stimulation in 20% and 53% of rats, respectively (rotigaptide versus control), resulting in reduction of arrhythmia score (3.2 versus 1.4, P=0.018), associated with the reduced magnetic resonance imaging parameters fractional anisotropy (fractional anisotropy: -5% versus -15%; P=0.062) and mean diffusivity (mean diffusivity: 2% versus 6%, P=0.042), and remodeling of the 3-dimensional laminar structure of the infarct border zone with reduction of the mean (16° versus 19°, P=0.013) and the dispersion (9° versus 12°, P=0.015) of the myofiber transverse angle. There was no change in ECG features, spontaneous arrhythmias, or mortality. Conclusions Enhancement of gap junctions function by rotigaptide administered during the early healing phase in reperfused infarction reduces later complexity of infarct scar morphology and programmed electrical stimulation-induced arrhythmias, and merits further exploration as a feasible and practicable intervention in the acute myocardial infarction management to reduce late arrhythmic risk.

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2 By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.