Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso–redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF.
In the presence of diabetes (DM), myocardial glucose uptake and glycolysis are impaired and the heart rapidly adapts to use exclusively fatty acids (FA) for ATP generation. This maladaptation is believed to play a key role in the development of a cardiomyopathy over time. Here, we show that stimulating myocardial nitric oxide synthase (NOS) activity is sufficient to alleviate myocardial metabolic inflexibility, improve energy metabolism and prevent LV dysfunction in DM by increasing myocardial insulin-independent glucose transport.
Methods
Myocardial-specific overexpression of GTP cyclohydrolase I (mGCH1) was used to increase both tetrahydrobiopterin (BH4) and NOS activity in cardiomyocytes. Diabetes mellitus (DM) was induced by multiple low-dose streptozotocin injections (vs sham). PCr/ATP ratio was measured in perfused hearts using 31P-MRS, glucose transport estimated by deoxy-glucose uptake, and oxygen consumption rate (OCR) of intact cardiomyocytes using a phosphorescent probe.
Results
As expected, sham-injected mGCH1 transgenic hearts had higher BH4 levels and constitutive NOS activity compared with WT. 12 weeks after DM induction, LV dysfunction developed in WT mice but not in mGCH1 mice, in the absence of changes in myocardial BH4 content and NOS activity in either group. WT diabetic hearts had a lower PCr/ATP ratio (1.32±0.1 vs 1.73±0.1, p<0.05, n=11 per group) and mitochondrial creatine kinase (CK) activity (1.56±0.1 AU vs 1.98±0.1 AU, p<0.005, n=10 per group) when compared with non-diabetic WT mice, consistent with impaired cardiac energetics. By contrast, PCr/ATP and CK activity were preserved in diabetic mGCH1 hearts in the absence of differences in myocardial mitochondrial content. Myocardial GCH1 overexpression was associated with a higher protein levels of the insulin-independent glucose transporter, GLUT-1 (p<0.05, n=12 per group), but no changes in GLUT-4 protein. Myocardial glucose transport was 40% higher in LV myocytes from mGCH1 diabetic mice when compared with WT diabetic mice. This was accompanied by increased myocardial glucose oxidation, as determined by OCR. Pre-incubation of myocytes with inhibitors of NOS-PKG signalling (L-NAME, 1 mmol/L or Rp8pCPT PET cGMP 10 µmol/L) or GLUT-1 (STF-31, 10 µmol/L,) abolished all differences between mGCH and WT diabetic hearts.
Conclusions
Our study reveals that a myocardial increase in BH4 and NOS activity is sufficient to maintain a favourable substrate utilisation and preserve cardiac mitochondrial function in the presence of DM. This work provides new insight into the potential metabolic triggers of diabetic cardiomyopathy and suggests exciting new targets for BH4-based therapeutics.
Abstract Background Hyperimmune convalescent COVID-19 plasma (CCP) containing anti-SARS-CoV-2 neutralizing antibodies (NAbs) was proposed as a therapeutic option for patients early in the new coronavirus disease pandemic. The efficacy of this therapy depends on the quantity of neutralizing antibodies (NAbs) in the CCP units, with titers ≥ 1:160 being recommended. The standard neutralizing tests (NTs) used for determining appropriate CCP donors are technically demanding and expensive and take several days. We explored whether they could be replaced by high-throughput serology tests and a set of available clinical data. Methods Our study included 1302 CCP donors after PCR-confirmed COVID-19 infection. To predict donors with high NAb titers, we built four (4) multiple logistic regression models evaluating the relationships of demographic data, COVID-19 symptoms, results of various serological testing, the period between disease and donation, and COVID-19 vaccination status. Results The analysis of the four models showed that the chemiluminescent microparticle assay (CMIA) for the quantitative determination of IgG Abs to the RBD of the S1 subunit of the SARS-CoV-2 spike protein was enough to predict the CCP units with a high NAb titer. CCP donors with respective results > 850 BAU/ml SARS-CoV-2 IgG had a high probability of attaining sufficient NAb titers. Including additional variables such as donor demographics, clinical symptoms, or time of donation into a particular predictive model did not significantly increase its sensitivity and specificity. Conclusion A simple quantitative serological determination of anti-SARS-CoV-2 antibodies alone is satisfactory for recruiting CCP donors with high titer NAbs.
Following acute myocardial infarction (AMI), monocytes are rapidly mobilised from the spleen to peripheral blood, from where they undergo transcriptional activation and infiltrate injured tissue, with potential to contribute to both injury and repair. The mechanism by which the injured myocardium signals splenic-monocyte mobilisation remains poorly understood. Recent work shows extracellular vesicles (EV, which carry proteins, microRNA/mRNA) are a means of rapid cell-to-cell communication, which, combined with knowledge of their composition and propensity to be taken up by other cells, suggests a possible role in signalling. Here we show that AMI results in a net increase in circulating endothelial cell (EC)-EV that induce splenic monocyte motility in vivo and cellular transcription.
Methods
Platelet-poor plasma was collected from patients with ST-segment elevation-AMI (STEMI) and mice subjected to AMI. EV were isolated by ultra-centrifugation and analysed for size/number by Nanoparticle Tracking Analysis, western blot (EV-markers: ALIX, TSG101, CD69, CD9 and Hsp70), ELISA for EC markers (CD31, ICAM-1, P-selectin, E-selectin and VCAM-1), electron microscopy and for EV-miRNAs. Human and mouse EC were used in vitro to evaluate EV release, injected into wild-type or CD68GFP+ naïve mice to assess bio-distribution, splenic-monocyte mobilisation, uptake by monocytes, cellular mRNA transcription and cell motility.
Results
Acutely (24 hours) after AMI there is a significant increase in circulating EV in humans (p<0.01) and mice (p<0.001) that later subsides. Plasma EV number correlates with myocardial injury in humans (R2=0.52, p<0.01). Plasma EV display EC-surface markers and show enrichment for vascular cell adhesion moleculae-1 (VCAM-1) in AMI (p<0.05). In vitro pro-inflammatory cytokines significantly increase EV production by EC, whereas ‘anti-inflammatory’ IL-4 and IL-6 had no effect. Inflammatory-EC-EV displayed significant enrichment of VCAM-1 (p<0.05). In-vitro labelled EC-EV accumulate in monocytes. Inflammatory-EC-EV significantly enhanced macrophage chemokineses (p<0.05) and chemotaxis to MCP-1 (p<0.05), a response that was abolished by pre-incubating EC-EV with an anti-VCAM-1 antibody (p<0.05). Injected labelled EC-EV accumulate in the spleen, interact with splenic monocytes and induce splenic-monocyte mobilisation and peripheral monocytosis in-vivo (p<0.01). Human plasma-EV show enrichment for 12 miRNAs in AMI, including EC-associated miR-126–3p/5p. miRNA-mRNA target gene prediction and functional enrichment analysis show roles for these miRNAs in the positive regulation of chemotaxis, cellular growth and proliferation. EC-EV significantly induced alterations in mRNA of motility genes by reducing PLEXIN-B2 (p<0.001), a negative regulator of motility and increasing ITGB2 (p<0.001) expression in monocytes.
In conclusion
(1) AMI surges plasma EV; (2) Plasma-EV protein composition is consistent with EC origin. (3) Injected EV localise to the spleen and (4) mobilise splenic monocytes. (5) In culture, EC increase EV release, enhance monocyte motility and (6) regulate genes that are important in cellular movement. These demonstrate a novel role for EC-derived EV in monocyte activation after AMI.
Abstract Breakthroughs in targeted KRAS therapeutics (KRASi) have the potential to transform the treatment landscape for several of the most common cancers including lung, colorectal, and pancreatic. Despite the recent approvals of KRASi and the anticipation of more to come, both the rate of patient response and the durability of these responses remain significant areas requiring improvement. Biomarkers that can predict response to KRASi and guide effective patient selection and drug combination strategies will be key to realizing the full potential of this emerging therapeutic field. While most biomarkers predominantly rely on a single analyte (e.g. KRAS mutation status), Genialis’ biomarkers are constructed using high-dimensional and/or multimodal data that capture the underlying biological complexity unique to each individual patient. Genialis' ResponderID™ is a machine learning-based biomarker discovery framework that models fundamental aspects of cancer biology to predict the clinical benefit based on the patient’s own biology. Here we report progress towards the development of a first-in-class, RNA-based biomarker, ResponderID™ KRAS, capable of stratifying KRAS G12C inhibitor response in lung cancer patients using RNA sequencing data. Trained on thousands of lung cancer samples, our biomarker models therapeutic response by unifying two core KRAS biologic axes, dependency and activation, to identify those patients most likely to respond. The performance characteristics of ResponderID™ KRAS thus far has been evaluated on a real world dataset of lung cancer patients treated with Sotorasib. ResponderID™ KRAS serves as an independent biomarker designed to inform clinical trial design, select for therapeutic efficacy, identify rational combination strategies, and expedite approvals across various therapeutic contexts. Citation Format: Josh Wheeler, Anže Lovše, Klemen Žiberna, Miha Štajdohar, Luka Ausec, Janez Kokošar, Daniel Pointing, Aditya Pai, Rafael Rosengarten, Mark Uhlik. ResponderID™ KRAS: Biology-driven machine learning to personalize KRAS inhibitor therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6446.
The association of the ABO blood group with COVID-19 disease has been confirmed by several studies, with the blood group A patients being more susceptible and prone to a more severe clinical course of the disease. Additionally, several authors also addressed the association of ABO-types and the levels of anti-SARS-CoV-2 antibodies in convalescents, mostly supporting a theory that the non-O blood group convalescents present with higher levels of anti-SARS-CoV-2 antibodies.Since previous findings were based on small convalescent cohorts, we quantified the anti-SARS-CoV-2 antibody levels in a total of 3187 convalescent plasma donors with three commercial serological and one standard neutralizing antibody test. The majority of donors had undergone a mild form of the disease and the median time of sampling was 66 days after diagnosis.None of the antibody quantitation results showed any significant association with the ABO blood group types. The same result was evident in the subgroup of vaccinated individuals (n = 370) and the subgroups when stratified according to post-COVID-19 periods (0-60, 60-120, and 120-180 days).In conclusion, we found no evidence to confirm that the ABO blood group types influence the level of SARS-CoV-2 antibody response in COVID-19 convalescent plasma donors.
In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype.Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes.In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31phosphorous magnetic resonance spectroscopy), and myocardial function.We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.
Background: At the beginning of the pandemic, COVID-19 convalescent plasma (CCP) containing anti-SARS-CoV-2 antibodies was suggested as a source of therapy. In the last three years, many trials have demonstrated the limited usefulness of CCP therapy. This led us to the hypothesis that CCP could contain other elements, along with the desired neutralizing antibodies, which could potentially prevent it from having a therapeutic effect, among them cytokines, chemokines, growth factors, clotting factors, and autoantibodies. Methods: In total, 39 cytokines were analyzed in the plasma of 190 blood donors, and further research focused on the levels of 23 different cytokines in CCP (sCD40L, eotaxin, FGF-2, FLT-3L, Fractalkine, GRO-α, IFNα2, IL-1β, IL-1RA, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17E, IP 10, MCP-1, MIP-1b, PDGF-AA, TGFα, TNFα, and TRAIL). Anti-SARS-CoV-2 antibodies and neutralizing antibodies were detected in CCP. Results: We found no significant differences between CCP taken within a maximum of 180 days from the onset of the first COVID-19 symptoms and the controls. We also made a comparison of the cytokine levels between the low neutralizing antibodies (<160) group and the high neutralizing antibodies (≥160) group and found there were no differences between the groups. Our research also showed no correlation either to levels of anti-SARS-CoV-2 IgG Ab or to the levels of neutralizing antibodies. There were also no significant changes in cytokine levels based on the period after the start of COVID-19 symptoms. Conclusions: No elements which could potentially be responsible for preventing CCP from having a therapeutic effect were found.
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