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Aims Recent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans. Methods and results Study 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide (O2.−) sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with O2.− production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM. Conclusions We demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin., Graphical Abstract Proposed mechanism of canagliflozin-induced improvement of myocardial redox state. Canagliflozin increases intracellular AMP/ATP ratio through inhibition of SGLT1, which can activate AMPK/NOS signalling and increase NO that suppresses pro-inflammatory signalling. AMPK activation also inhibits activation of Rac1 and membrane translocation of Rac1 and p47phox, which decrease NADPH oxidase activity and superoxide (O2.−) production, attenuates inflammatory and apoptotic pathways and increasing the bioavailability of tetrahydrobiopterin (BH4), a key factor for NOS coupling.

Introduction Myocardial creatine kinase (CK) activity represents an important metabolic reserve: it correlates closely with contractile reserve and post-ischaemic function, keeps cytosolic [ADP] low, and optimises the free energy for ATP hydrolysis. It may also contribute to the transition to failure in the heart hypertrophied by chronic haemodynamic overload; modest up-regulation has been shown to be cardio-protective. Total CK activity measurement requires chemical freeze-and-extract methods, which destroy tissue, precluding repeated measures. To date, non-invasive assessments of human myocardial CK flux (calculated as kf × [PCr], where kf is the pseudo-first-order forward rate constant measured by 31P-magnetic resonance spectroscopy (MRS) have not assessed creatine content or total CK activity. Thus we aimed to validate kf measurement against total CK activity and investigate predictors of CK activity. Methods 39 subjects (median age 71, range 43–84) undergoing clinically indicated cardiac surgery had CK total activity measured from LV biopsy. 31 had severe AS (10 with impaired LVEF); 2 had severe primary MR; 5 had severe mitral stenosis (2 with impaired LVEF) and 1 had an LA mass. 35 of 39 contributed triplicate datasets: CK total activity, kf (31P-MRS TRiST sequence at 3T) and LV volumes (cine-MRI, 3T Siemens). 27 had severe AS (8 with impaired LVEF); other groups were the same. Exclusion criteria were prior myocardial infarction and flow-limiting coronary disease. Flash-frozen LV biopsies obtained within 15 min of cardiopulmonary bypass were analysed for CK total activity, total creatine, and citrate synthase (CS) activity (a marker of oxidative phosphorylation capacity). Results Multiple, novel correlations were observed between CK total activity (IU/mg protein) and CS activity (r=0.87, p=9e-13), total creatine (r=0.59, p=8e-5), kf (r=0.42, p=0.013), total creatine × kf (r=0.64, p=4e-5), LVESVi (r=−0.52, p=8e-4), LVEF (r=0.48, p=0.002) and LVMi (r=−0.42, p=0.009) (Panels A-E) (LVEDVi and non-indexed counterparts were also significant correlates.) The most predictive linear regression model incorporating elements of the CK rate equation included total creatine (nmol/mg protein), kf (/s), and kf × LVESVi (ml/m2) (adjusted R2=0.56, beta=0.426, 0.618, −1.968 and p=0.001, 5e-5, 0.003 respectively, Panel F). Figure 1 Conclusions These results are the first evidence of agreement between non-invasive estimates of human cardiac CK activity (kf) and freeze-extracted chemical methods. The key original finding is that it is feasible to attempt to predict CK capacity in vivo by using a combination of techniques (creatine by 1H MRS, CK kf by 31P-MRS and LVESVi by cine imaging). The key insight is that CK capacity is best estimated not simply from what the rate equation would predict (creatine and kf), but that other factors relating to failing contractility reduce CK activity independently from creatine content and enzyme kf. Acknowledgement/Funding British Heart Foundation Clinical Research Training Fellowship (FS/15/80/31803) and Programme Grant (RG/18/12/34040).

Introduction The transition to systolic failure in severe aortic stenosis (AS) increases mortality. There are currently no reliable markers of transition, and the guideline LVEF Methods 19 patients with severe AS, LVEF ≥55% (AS-pEF, mean±SD LVEF 63±5%, mean gradient 48±14 mmHg) and 10 with severe AS, LVEF Results When compared to controls, both CK total activity and CS activity were lower in AS-pEF (by 27% and 23% respectively, both p AS-rEF was associated with further reduction in both CK and CS activities (by 32% and 22% respectively, both p When median mitochondria-sarcomere ATP diffusion distances were plotted against CK total activity a strong positive correlation was observed (r=0.86, p=0.003, Panel F). This suggests a compensatory reduction in diffusion distance develops when CK activity falls. Conclusions Transition to failure in severe AS is associated with lower oxidative capacity and maximal ATP delivery capacity through CK. Despite compensatory falls in ATP diffusion distance and altered CK isozyme expression, these changes may underlie susceptibility to EF decline in AS. Acknowledgement/Funding British Heart Foundation Clinical Research Training Fellowship (FS/15/80/31803) and Programme Grant (RG/18/12/34040).

Background Wnt5a is a non-canonical Wnt ligand with potential vascular effects, but its mechanistic role in atherosclerosis progression and the underlying downstream mechanisms are poorly explored. Purpose To address the hypothesis that Wnt5a induces vascular NADPH-oxidases activity, endothelial dysfunction and detrimental downstream redox signalling which could propagate atherosclerosis in humans. Methods Study 1 included 70 patients with coronary artery disease (CAD) versus age- and sex-matched non-CAD controls. Study 2 included 1,003 CAD patients undergoing cardiac surgery; internal mammary artery (IMA) and saphenous vein (SV) segments were harvested and used for ex vivo experiments. Study 3 included 68 individuals undergoing two cardiac computed tomography scans 3–5 years apart; calcified plaque burden was assessed by coronary calcium score (CCS). Superoxide (O2·−) generation was measured by lucigenin chemiluminescence with NADPH 100μM stimulation as indicator of NADPH-oxidases activity. Activation of Rac1, a key NADPH-oxidases subunit, was evaluated by a commercially available kit. Primary vascular smooth muscle cells (VSMCs) and HeLa cells were used for in vitro experiments. Circulating Wnt5a and Sfrp5 (a Wnt5a antagonist) were measured by ELISA in fasting plasma samples. Results In Study 1, the presence of CAD was independently linked with increased circulating Wnt5a bioavailability (A), which was, in turn, associated with increased IMA NADPH-oxidases activity in Study 2 (B). Recombinant Wnt5a directly stimulated NADPH-oxidases activity (C) via Rac1 activation (not shown) in human IMA, while inducing endothelial dysfunction evidenced by impaired SV endothelium-dependent acetylcholine (Ach) vasorelaxations (D). Transcriptomic analysis in Wnt5a-treated primary VSMCs versus controls identified USP17, a deubiquitinating enzyme implicated in Rac1 activation, as the top differentially regulated hit (not shown). Indeed, Wnt5a stimulated USP17 upregulation in VSMCs which was reversed by PEGylated superoxide dismutase (peg-SOD) 300U/mL (E), suggesting a redox sensitive effect. USP17 knockdown abolished the ability of Wnt5a to induce Rac1 activation in HeLa cells (F). At a clinical level, plasma Wnt5a was a predictor of plaque progression (defined as ΔCCS≥1, G) and new onset calcification (H) in Study 3. Conclusions We demonstrate for the first time that Wnt5a is elevated in CAD and causally associated with increased vascular oxidative stress and endothelial dysfunction in humans. We further reveal USP17 to be a novel, previously undescribed, link between Wnt5a, Rac1 activation and NADPH-oxidase activity induction in humans. We finally propose that circulating Wnt5a may have a clinically relevant role in predicting atherosclerosis progression. Our findings identify Wnt5a as rational therapeutic target in vascular disease. Acknowledgement/Funding British Heart Foundation; Alexandros S Onassis Public Benefit Foundation

Background NADPH oxidase activity is a major source of superoxide anions (O2·−) in the human myocardium, linked with adverse short-term clinical outcomes after cardiac surgery, but its prognostic value for the long-term clinical outcome of these patients is unknown. Purpose To explore the association between NADPH oxidase activity in the human myocardium and the clinical outcome of patients undergoing cardiac surgery. Methods A total of 216 patients from the Oxford Heart Vessels and Fat (OxHVF) cohort were enrolled. Myocardial tissue samples were collected during cardiac surgery and used for RNA isolation and myocardial O2·− quantification using lucigenin-enhanced chemiluminescence (5μmol/L). The specific contribution of NADPH oxidase activity to myocardial O2·− production was quantified as the Vas287021 (40 μmol/L)-inhibitable signal following NADPH (100μmol/L) stimulation (Vas2870 is a NADPH oxidases inhibitor). Malonyldialdehyde and hsCRP levels were measured in plasma isolated from peripheral blood collected pre-operatively. Patients were followed-up for a mean of 2.9±1.4 years for the primary endpoint of cardiovascular mortality. Results Myocardial NADPH oxidase activity was not correlated with systemic oxidative stress (evidenced by malonyldialdehyde, panel A) or systemic inflammation (reflected by hsCRP levels, panel B). NADPH oxidase activity was significantly higher in the cardiovascular death group (panel C) and was associated with cardiovascular (HR [95% CI]: 1.53 [1.10–2.13], p=0.01 per SD increase) but not all-cause mortality (HR [95% CI]: 1.32 [0.96–1.80], p=0.08 per SD increase). This association remained significant after adjustment for EuroscoreII (HR [95% CI]: 1.80 [1.14–2.82], p=0.01 per SD increase). For an optimal cut-off value of 7,920 RLU/sec/mg tissue, patient survival was significantly poorer in the high vs. low NADPH oxidase activity subgroup (panel D). Adding NADPH oxidase activity to the baseline clinical model significantly improved model's performance (panel E). Interestingly, myocardial NOX gene expression levels didn't show any significance in mortality prediction, highlighting the fact that NADPH oxidase activity, rather than enzyme levels, is the key outcomes regulator. Conclusion Increased NADPH oxidase activity in the human myocardium independently predicts high cardiovascular mortality in patients with advanced coronary atherosclerosis. Targeting NADPH oxidase in the human heart may be a rational therapeutic approach. Acknowledgement/Funding BHF, EPSRC

Experimental evidence suggests that telomere length (TL) is shortened by oxidative DNA damage, reflecting biological aging. We explore the value of blood (BTL) and vascular TL (VTL) as biomarkers of systemic/vascular oxidative stress in humans and test the clinical predictive value of BTL in acute myocardial infarction (AMI).In a prospective cohort of 290 patients surviving recent AMI, BTL measured on admission was a strong predictor of all-cause [hazard ratio (HR) [95% confidence interval (CI)]: 3.21 [1.46-7.06], P = 0.004] and cardiovascular mortality (HR [95% CI]: 3.96 [1.65-9.53], P = 0.002) 1 year after AMI (for comparisons of short vs. long BTL, as defined by a T/S ratio cut-off of 0.916, calculated using receiver operating characteristic analysis; P adjusted for age and other predictors). To explore the biological meaning of these findings, BTL was quantified in 727 consecutive patients undergoing coronary artery bypass grafting (CABG), and superoxide (O2.-) was measured in peripheral blood mononuclear cells (PBMNC). VTL/vascular O2.- were quantified in saphenous vein (SV) and mammary artery (IMA) segments. Patients were genotyped for functional genetic polymorphisms in P22ph°x (activating NADPH-oxidases) and vascular smooth muscle cells (VSMC) selected by genotype were cultured from vascular tissue. Short BTL was associated with high O2.- in PBMNC (P = 0.04) but not in vessels, whereas VTL was related to O2.- in IMA (ρ = -0.49, P = 0.004) and SV (ρ = -0.52, P = 0.01). Angiotensin II (AngII) incubation of VSMC (30 days), as a means of stimulating NADPH-oxidases, increased O2.- and reduced TL in carriers of the high-responsiveness P22ph°x alleles (P = 0.007).BTL predicts cardiovascular outcomes post-AMI, independently of age, whereas VTL is a tissue-specific (rather than a global) biomarker of vascular oxidative stress. The lack of a strong association between BTL and VTL reveals the importance of systemic vs. vascular factors in determining clinical outcomes after AMI.

Background: Internal mammary artery (IMA) is used as a graft in bypass surgery (CABG), but it is unclear whether it should be harvested with or without its perivascular adipose tissue (PVAT). Given that PVAT produces the antiatherogenic adipokine adiponectin (AdN), we examined the role of AdN as a signalling molecule between PVAT and IMA grafts. Methods: In Study 1, IMA segments were obtained from 364 patients undergoing CABG and used to determine NADPH-oxidase activity by lucigenin chemiluminescence (+/-NADPH 100μM). Mesothoracic (Mt-AT), subcutaneous (Sc-AT) and PVAT were obtained for gene expression studies. In Study 2, IMA segments from 17 patients were exposed ex vivo to AdN (10 μg/ml) for 6 hours ± wortmannin (a PI3K/Akt inhibitor); changes to NADPH-stimulated and Vas2870 (specific NADPH-oxidase inhibitor)-inhibitable O2- as well as the activation and membrane translocation of Rac1 (NADPH-oxidase subunit responsible for its activation) were determined. Results: Elevated circulating AdN was related with reduced NADPH-stimulated O2- (A). Increased expression of ADIPOQ in Mt- and Sc-AT was related with reduced NADPH-oxidase activity, however in PVAT it was associated with increased NADPH-stimulated O2-(B). AdN directly suppressed NADPH-stimulated (C) and Vas2870-inihibitable (D) O2- in the IMA. This was due to reduced activation (E)/membrane translocation (F) of Rac1, which was reversed by wortmannin. ![Figure][1] Conclusions: We demonstrate for the first time in humans, that AdN directly suppresses NADPH-oxidase through an Akt-mediated reduction of Rac1 activation and translocation to the membrane. The observed increase of AdN expression in PVAT in the presence of high oxidative stress may represent a novel, local defence mechanism of IMA against oxidative stress, that warrants further investigation. [1]: pending:yes