Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2.

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.

Echocardiographic evaluation of diastolic function in mouse models of heart disease.

BACKGROUND: Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice. METHODS AND RESULTS: We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate. CONCLUSION: These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms.

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

Extracellular matrix secretion by cardiac fibroblasts: role of microRNA-29b and microRNA-30c.

RATIONALE: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. OBJECTIVE: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. METHODS AND RESULTS: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-ß. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre-miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre-miR-29 conditioned medium, whereas pre-miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. CONCLUSIONS: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.

Novel aspects of ROS signalling in heart failure.

Heart failure and many of the conditions that predispose to heart failure are associated with oxidative stress. This is considered to be important in the pathophysiology of the condition but clinical trials of antioxidant approaches to prevent cardiovascular morbidity and mortality have been unsuccessful. Part of the reason for this may be the failure to appreciate the complexity of the effects of reactive oxygen species. At one extreme, excessive oxidative stress damages membranes, proteins and DNA but lower levels of reactive oxygen species may exert much more subtle and specific regulatory effects (termed redox signalling), even on physiological signalling pathways. In this article, we review our current understanding of the roles of such redox signalling pathways in the pathophysiology of heart failure, including effects on cardiomyocyte hypertrophy signalling, excitation-contraction coupling, arrhythmia, cell viability and energetics. Reactive oxygen species generated by NADPH oxidase proteins appear to be especially important in redox signalling. The delineation of specific redox-sensitive pathways and mechanisms that contribute to different components of the failing heart phenotype may facilitate the development of newer targeted therapies as opposed to the failed general antioxidant approaches of the past.

The regulation of cardiac intermediary metabolism by NADPH oxidases.

NADPH oxidases (NOXs), enzymes whose primary function is to generate reactive oxygen species, are important regulators of the heart's physiological function and response to pathological insults. The role of NOX-driven redox signalling in pathophysiological myocardial remodelling, including processes such as interstitial fibrosis, contractile dysfunction, cellular hypertrophy, and cell survival, is well recognized. While the NOX2 isoform promotes many detrimental effects, the NOX4 isoform has attracted considerable attention as a driver of adaptive stress responses both during pathology and under physiological states such as exercise. Recent studies have begun to define some of the NOX4-modulated mechanisms that may underlie these adaptive responses. In particular, novel functions of NOX4 in driving cellular metabolic changes have emerged. Alterations in cellular metabolism are a recognized hallmark of the heart's response to physiological and pathological stresses. In this review, we highlight the emerging roles of NOX enzymes as important modulators of cellular intermediary metabolism in the heart, linking stress responses not only to myocardial energetics but also other functions. The novel interplay of NOX-modulated redox signalling pathways and intermediary metabolism in the heart is unravelling a new aspect of the fascinating biology of these enzymes which will inform a better understanding of how they drive adaptive responses. We also discuss the implications of these new findings for therapeutic approaches that target metabolism in cardiac disease.

The nexus between redox state and intermediary metabolism.

Reactive oxygen species (ROS) are not just a by-product of cellular metabolic processes but act as signalling molecules that regulate both physiological and pathophysiological processes. A close connection exists in cells between redox homeostasis and cellular metabolism. In this review, we describe how intracellular redox state and glycolytic intermediary metabolism are closely coupled. On the one hand, ROS signalling can control glycolytic intermediary metabolism by direct regulation of the activity of key metabolic enzymes and indirect regulation via redox-sensitive transcription factors. On the other hand, metabolic adaptation and reprogramming in response to physiological or pathological stimuli regulate intracellular redox balance, through mechanisms such as the generation of reducing equivalents. We also discuss the impact of these intermediary metabolism-redox circuits in physiological and disease settings across different tissues. A better understanding of the mechanisms regulating these intermediary metabolism-redox circuits will be crucial to the development of novel therapeutic strategies.

First-Phase Ejection Fraction, a Measure of Preclinical Heart Failure, Is Strongly Associated With Increased Mortality in Patients With COVID-19.

Presence of heart failure is associated with a poor prognosis in patients with coronavirus disease 2019 (COVID-19). The aim of the present study was to examine whether first-phase ejection fraction (EF1), the ejection fraction measured in early systole up to the time of peak aortic velocity, a sensitive measure of preclinical heart failure, is associated with survival in patients hospitalized with COVID-19. A retrospective outcome study was performed in patients hospitalized with COVID-19 who underwent echocardiography (n=380) at the West Branch of the Union Hospital, Wuhan, China and in patients admitted to King's Health Partners in South London, United Kingdom. Association of EF1 with survival was performed using Cox proportional hazards regression. EF1 was compared in patients with COVID-19 and in historical controls with similar comorbidities (n=266) who had undergone echocardiography before the COVID-19 pandemic. In patients with COVID-19, EF1 was a strong predictor of survival in each patient group (Wuhan and London). In the combined group, EF1 was a stronger predictor of survival than other clinical, laboratory, and echocardiographic characteristics including age, comorbidities, and biochemical markers. A cutoff value of 25% for EF1 gave a hazard ratio of 5.23 ([95% CI, 2.85-9.60]; P<0.001) unadjusted and 4.83 ([95% CI, 2.35-9.95], P<0.001) when adjusted for demographics, comorbidities, hs-cTnI (high-sensitive cardiac troponin), and CRP (C-reactive protein). EF1 was similar in patients with and without COVID-19 (23.2±7.3 versus 22.0±7.6%, P=0.092, adjusted for prevalence of risk factors and comorbidities). Impaired EF1 is strongly associated with mortality in COVID-19 and probably reflects preexisting, preclinical heart failure.

Drug treatment effects on outcomes in heart failure with preserved ejection fraction: a systematic review and meta-analysis.

BACKGROUND: Clinical drug trials in patients with heart failure and preserved ejection fraction have failed to demonstrate improvements in mortality. METHODS: We systematically searched Medline, Embase and the Cochrane Central Register of Controlled Trials for randomised controlled trials (RCT) assessing pharmacological treatments in patients with heart failure with left ventricular (LV) ejection fraction≥40% from January 1996 to May 2016. The primary efficacy outcome was all-cause mortality. Secondary outcomes were cardiovascular mortality, heart failure hospitalisation, exercise capacity (6-min walk distance, exercise duration, VO2 max), quality of life and biomarkers (B-type natriuretic peptide, N-terminal pro-B-type natriuretic peptide). Random-effects models were used to estimate pooled relative risks (RR) for the binary outcomes, and weighted mean differences for continuous outcomes, with 95% CI. RESULTS: We included data from 25 RCTs comprising data for 18101 patients. All-cause mortality was reduced with beta-blocker therapy compared with placebo (RR: 0.78, 95%CI 0.65 to 0.94, p=0.008). There was no effect seen with ACE inhibitors, aldosterone receptor blockers, mineralocorticoid receptor antagonists and other drug classes, compared with placebo. Similar results were observed for cardiovascular mortality. No single drug class reduced heart failure hospitalisation compared with placebo. CONCLUSION: The efficacy of treatments in patients with heart failure and an LV ejection fraction≥40% differ depending on the type of therapy, with beta-blockers demonstrating reductions in all-cause and cardiovascular mortality. Further trials are warranted to confirm treatment effects of beta-blockers in this patient group.

Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise.

Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise.

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.

Reporting trends of randomised controlled trials in heart failure with preserved ejection fraction: a systematic review.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) causes significant cardiovascular morbidity and mortality. Current consensus guidelines reflect the neutral results from randomised controlled trials (RCTs). Adequate trial reporting is a fundamental requirement before concluding on RCT intervention efficacy and is necessary for accurate meta-analysis and to provide insight into future trial design. The Consolidated Standards of Reporting Trials (CONSORT) 2010 statement provides a framework for complete trial reporting. Reporting quality of HFpEF RCTs has not been previously assessed, and this represents an important validation of reporting qualities to date. OBJECTIVES: The aim was to systematically identify RCTs investigating the efficacy of pharmacological therapies in HFpEF and to assess the quality of reporting using the CONSORT 2010 statement. METHODS: MEDLINE, EMBASE and CENTRAL databases were searched from January 1996 to November 2015, with RCTs assessing pharmacological therapies on clinical outcomes in HFpEF patients included. The quality of reporting was assessed against the CONSORT 2010 checklist. RESULTS: A total of 33 RCTs were included. The mean CONSORT score was 55.4% (SD 17.2%). The CONSORT score was strongly correlated with journal impact factor (r=0.53, p=0.003) and publication year (r=0.50, p=0.003). Articles published after the introduction of CONSORT 2010 statement had a significantly higher mean score compared with those published before (64% vs 50%, p=0.02). CONCLUSIONS: Although the CONSORT score has increased with time, a significant proportion of HFpEF RCTs showed inadequate reporting standards. The level of adherence to CONSORT criteria could have an impact on the validity of trials and hence the interpretation of intervention efficacy. We recommend improving compliance with the CONSORT statement for future RCTs.

Endoplasmic reticulum stress and Nox-mediated reactive oxygen species signaling in the peripheral vasculature: potential role in hypertension.

SIGNIFICANCE: Reactive oxygen species (ROS) are produced during normal endoplasmic reticulum (ER) metabolism. There is accumulating evidence showing that under stress conditions such as ER stress, ROS production is increased via enzymes of the NADPH oxidase (Nox) family, especially via the Nox2 and Nox4 isoforms, which are involved in the regulation of blood pressure. Hypertension is a major contributor to cardiovascular and renal disease, and it has a complex pathophysiology involving the heart, kidney, brain, vessels, and immune system. ER stress activates the unfolded protein response (UPR) signaling pathway that has prosurvival and proapoptotic components. RECENT ADVANCES: Here, we summarize the evidence regarding the association of Nox enzymes and ER stress, and its potential contribution in the setting of hypertension, including the role of other conditions that can lead to hypertension (e.g., insulin resistance and diabetes). CRITICAL ISSUES: A better understanding of this association is currently of great interest, as it will provide further insights into the cellular mechanisms that can drive the ER stress-induced adaptive versus maladaptive pathways linked to hypertension and other cardiovascular conditions. More needs to be learnt about the precise signaling regulation of Nox(es) and ER stress in the cardiovascular system. FUTURE DIRECTIONS: The development of specific approaches that target individual Nox isoforms and the UPR signaling pathway may be important for the achievement of therapeutic efficacy in hypertension.