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We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Aims Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial. Methods and results We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71–76%, n = 46 paired samples, P, Graphical Abstract

Supplemental Digital Content is available in the text., Background: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. Methods: Bone marrow–derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr −/− mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. Results: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow–derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr −/− mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow–derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. Conclusions: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Introduction Diabetes mellitus (DM) is an important risk factor for atrial fibrillation (AF) – the most common heart rhythm disorder. However, the mechanisms underlying this association are poorly understood. In addition, patients with DM often have comorbidities, such as obesity, hypertension, dyslipidaemia, that are also independently associated with increased risk for AF. The aim of this project was to explore whether prolonged hyperglycaemia alone is sufficient to increase the risk of AF. Methods Multiple low-dose (50 mg/kg over 5 days) intraperitoneal injections of streptozotocin were used to induce DM in C57BL/6 mice. After 12 weeks of DM, left ventricular (LV) systolic and diastolic function was characterised using echocardiography. In vivo atrial electrophysiological properties and arrhythmia inducibility were assessed using transoesophageal atrial pacing. Atrial conduction time and action potential duration (APD) and conduction velocity were measured by optical mapping of isolated atria. Results Diabetic mice had significantly higher probability of in vivo AF induction (15±3% vs. 6±1%, in controls, p=0.005, n=24–26/group), atrial ECG conduction abnormalities (longer PQ interval in diabetic mice: 44±1 ms vs. 39±1 ms in controls, p=0.001, n=24–26/group), but without any changes in the P wave duration, RR, QRS or QT intervals. Diabetic mice developed modest LV diastolic dysfunction (tissue Doppler E’/A’ 1.02±0.05 vs. 1.20±0.06 in controls, p=0.04, n=11–12/group), but no LV systolic dysfunction. In addition, diabetic mice also had larger relative LA size (LA area/body weight 0.28±0.01 mm2/g vs. 0.23±0.01 mm2/g in controls, p=0.001, n=11–12/group). Optical mapping revealed a two-fold greater conduction time in diabetic left atria (LA) (39±3 ms vs. 22±2 ms in controls, p=0.0006, n=6/group), without differences in the right atrial (RA) conduction time. The APD was not significantly different in RA or LA. The regional analysis of optical mapping recordings demonstrated a significant decrease in the conduction velocity in the medial part of the RA resulting in increased conduction wavefront roughness in the LA. Finally, the medial part of the diabetic RA also had a significantly higher total collagen content (5.5±0.5 µg/mg vs. 9.1±0.6 µg/mg in controls, p=0.0002, n=9–11/group), and a decrease in the connexin 43 levels (0.92±0.05 vs. 0.66±0.4 in controls, p=0.001, n=7–8/group), with no significant changes in other parts of the atria. Conclusions Prolonged hyperglycaemia alone is sufficient to cause pathological remodelling making atria more susceptible for AF. Diabetic hearts also have modest LV diastolic dysfunction, relative LA enlargement, but no LV systolic dysfunction. In this study, we provide evidence of a novel mechanism where localized (rather than global) atrial fibrosis in a critical area causes a focal conduction defect and predisposes diabetic mice to AF.

Background and Aims: BH4 is successfully used in the clinic for inherited BH4 deficiency and BH4-responsive phenylketonuria. In recent years, BH4 supplementation has also drawn attention as a therapy for various nitric oxide synthase (NOS)-related cardiovascular pathologies. By genetic intervention, we have been able to increase cardiac intracellular BH4 levels, modify cardiac metabolism and prevent heart dysfunction in a murine model of diabetic cardiomyopathy. The aim of this study was to assess the efficacy of an oral BH4 preparation in our animal model before translating the treatment into diabetic patients. In particular, we tested whether BH4 oral supplementation would be sufficient to increase BH4/NO levels in cardiac tissue and if this would protect the diabetic heart. Results and Methods: Diabetes was induced by streptozotocin injections over 5 consecutive days in WT mice. After 12 weeks of diabetes, mice were fed with either placebo or BH4 diet (200 mg/kg/day) for an additional 6 weeks. At the end of this period, the group of diabetic mice treated with BH4 showed a significant increase of this biopterin in the heart (9.4 ± 1.3 pmol/mg protein vs 5.8 ± 0.8 pmol/mg in non-supplemented WT. P=0.034. N=8 hearts per group), as well as an increase in the activity of NOS (0.6 ± 0.11 vs 0.2 ± 0.05 % citrulline conversion. P=0.009). WT diabetic mice showed impaired diastolic function as indicated by tissue Doppler analysis (Lower E’/A’ ratio, P Conclusion Oral BH4 was sufficient to increase the level of this biopterin and NO availability in cardiac tissue. As a result, cardiac function was preserved in a mouse model of diabetic cardiomyopathy. These results have prompted us to test the effects of BH4 on cardiac metabolism and function in diabetic patients. Conflict of interest None

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and

Medium-chain length polyhydroxyalkanoates (MCL-PHAs) have demonstrated exceptional properties for cardiac tissue engineering (CTE) applications. Despite prior work on MCL-PHA/polycaprolactone (PCL) blends, optimal scaffold production and use as an alternative delivery route for controlled release of seeded cardiac progenitor cells (CPCs) in CTE applications in vivo has been lacking. We present herein applicability of MCL-PHA/PCL (95/5 wt %) blends fabricated as thin films with an improved performance compared to the neat MCL-PHA. Polymer characterization confirmed the chemical structure and composition of the synthesized scaffolds, while thermal, wettability, and mechanical properties were also investigated and compared in neat and porous counterparts. In vitro cytocompatibility studies were performed using perfluorocrown-ether-nanoparticle-labeled murine CPCs and studied using confocal microscopy and 19F magnetic resonance spectroscopy and magnetic resonance imaging (MRI). Seeded scaffolds were implanted and studied in the postmortem murine heart in situ and in two additional C57BL/6 mice in vivo (using single-layered and double-layered scaffolds) and imaged immediately after and at 7 days postimplantation. Superior MCL-PHA/PCL scaffold performance has been demonstrated compared to MCL-PHA through experimental comparisons of (a) morphological data using scanning electron microscopy and (b) contact angle measurements attesting to improved CPC adhesion, (c) in vitro confocal microscopy showing increased SC proliferative capacity, and (d) mechanical testing that elicited good overall responses. In vitro MRI results justify the increased seeding density, increased in vitro MRI signal, and improved MRI visibility in vivo, in the double-layered compared to the single-layered scaffolds. Histological evaluations [bright-field, cytoplasmic (Atto647) and nuclear (4′,6-diamidino-2-phenylindole) stains] performed in conjunction with confocal microscopy imaging attest to CPC binding within the scaffold, subsequent release and migration to the neighboring myocardium, and increased retention in the murine myocardium in the case of the double-layered scaffold. Thus, MCL-PHA/PCL blends possess tremendous potential for controlled delivery of CPCs and for maximizing possible regeneration in myocardial infarction.

Introduction 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 31 P-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 Myocardial GCH1 overexpression was associated with a higher protein levels of the insulin-independent glucose transporter, GLUT-1 (p 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.

An essential factor for the production of nitric oxide by nitric oxide synthase 1 (NOS1), major modulator of cardiac function, is the cofactor tetrahydrobiopterin (BH4). BH4 is regulated by GTP cyclohydrolase 1, the rate-limiting enzyme in BH4 biosynthesis which catalyses the formation of dihydroneopterin 3′triphosfate from GTP, producing BH4 after two further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. However, there are other essential factors involved in the regulation of NOS1 activity, such as protein inhibitor of NOS1 (PIN), calmodulin, heat shock protein 90 and NOS interacting protein. All these molecules have never been analysed in human non-ischemic dilated hearts (DCM). In this study we demonstrated that the upregulation of cardiac NOS1 is not accompanied by increased NOS1 activity in DCM, partly due to the elevated PIN levels and not because of alterations in biopterin biosynthesis. Notably, the PIN concentration was significantly associated with impaired ventricular function, highlighting the importance of this NOS1 activity inhibitor in Ca2+ homeostasis. These results take a central role in the current list of targets for future studies focused on the complex cardiac dysfunction processes through more efficient harnessing of NOS1 signalling.

PURPOSE: Diabetes can impact on cardiovascular health by causing a distinct condition termed "diabetic cardiomyopathy". Its characteristic left ventricular (LV) diastolic dysfunction has been associated with interstitial fibrosis, reduced NO availability, and abnormal calcium handling. However, the early triggers and the underlying cellular mechanisms remain unknown. Here, we investigate changes in vascular and myocardial reactive oxygen species (ROS) and NO availability in a murine model of type 1 diabetes, and evaluate potential beneficial effects of inducing a myocardial-specific increase in the NOS cofactor tetrahydrobiopterin (BH4) on the development of LV dysfunction. Methods: Diabetes was induced in male mice by daily intraperitoneal streptozotocin (STZ) injection (43mg/kg, 5 consecutive days). To augment myocardial BH4 and increase NOS activity, transgenic mice were generated with myocardial-specific overexpression of the rate-limiting enzyme for BH4 synthesis, GTP cyclohydrolase 1 (mGCH1 Tg). Vascular function in isolated aortas was evaluated by isometric tension studies (myograph), NOS activity and biopterins by HPLC, and superoxide production by lucigenin-enhanced chemiluminescence. High-resolution echocardiography was used to assess LV function. Results: After 12 weeks of diabetes, WT and mGCH1 Tg mice showed impaired aortic endothelium-dependent vasodilatation, in association with increased superoxide production and reduced BH4 bioavailability (n=6-10 per group). By contrast, diabetic LV homogenates showed no increase in superoxide generation or reduced BH4:BH2 ratio and no reduction in NOS activity (n=9-12 per group). Nevertheless, in vivo echocardiography revealed significant LV diastolic dysfunction in WT diabetic mice, which was prevented in mGCH1 Tg mice (E'/A' diabetic vs control: 1.52±0.08 vs 1.53±0.08 in mGCH1 Tg; 0.89±0.07 vs 1.35±0.06 in WT, n=9 per group, P

Background The management of atrial fibrillation remains a challenge. This condition remodels atrial electrical properties, which promote resistance to treatment. Although remodelling has long been a therapeutic target in atrial fibrillation, its causes remain incompletely understood. We aimed to evaluate the role of miR-31-dependent reduction in dystrophin and neuronal nitric oxide synthase (nNOS, also known as NOS1) on atrial electrical properties and atrial fibrillation inducibility. Methods We recruited 258 patients (209 patients in sinus rhythm and 49 with permanent atrial fibrillation) from the John Radcliffe Hospital, Oxford, UK; written informed consent was obtained from each participant. We also used a goat model of pacing-induced atrial fibrillation (24 with atrial fibrillation vs 20 controls in normal sinus rythm) and nNos-knock-out mice (n=28 compared with 27 wild-type littermates). Gene expression of miR-31, dystrophin, and nNOS was assessed by quantitative RT-PCR; protein content was measured by immunoblotting; NOS activity was evaluated with high-performance liquid chromatography; action potential duration (APD) and rate dependent adaptation were assessed by single-cell patch-clamping, and atrial fibrillation inducibility was evaluated by transoesophageal atrial burst stimulation. Findings We found that atrial-specific upregulation of miR-31 in human atrial fibrillation caused dystrophin ( DYS ) translational repression and accelerated mRNA degradation of nNOS leading to a profound reduction in atrial DYS and nNOS protein content and in nitric oxide availability. In human atrial myocytes obtained from patients in sinus rhythm, nNOS inhibition was sufficient to recapitulate hallmark features of remodelling induced by atrial fibrillation, such as shortening of APD and loss of APD rate-dependency, but had no effect in patients with atrial fibrillation. In mice, nNos gene deletion or inhibition shortened atrial APD and increased atrial fibrillation inducibility in vivo. Inhibition of miR-31 in human atrial fibrillation recovered DYS and nNOS, and normalised APD and APD rate-dependency. Prevention of miR-31 binding to nNOS 3′UTR recovered both nNOS protein and gene expression but had no effect on the DYS protein or mRNA level (consistent with the mRNA degradation of nNOS by miR-31). Prevention of miR-31 binding to DYS 3′UTR increased DYS protein but not mRNA is consistent with translation repression of DYS by miR-31; recovery of DYS protein increased nNOS protein but not mRNA in keeping with a stabilising effect of DYS on nNOS protein. In goats, a reduction in dystrophin and nNOS protein content was associated with upregulation of miR-31 in the atria but not in the ventricles. Interpretation The findings suggest that atrial-specific upregulation of miR-31 in human atrial fibrillation is a key mechanism causing atrial loss of dystrophin and nNOS; this loss leads to the electrical phenotype induced by atrial fibrillation. Funding British Heart Foundation (BHF) Programme grant (for BC and XL), BHF Centre of Excellence in Oxford (SR), Leducq Foundation (in part for BC and SR), the European Union's seventh Framework Programme Grant Agree.

The role of nitric oxide synthase 1 (NOS1) as a major modulator of cardiac function has been extensively studied in experimental models; however, its role in human ischemic cardiomyopathy (ICM) has never been analysed. Thus, the objectives of this work are to study NOS1 and NOS-related counterparts involved in regulating physiological function of myocyte, to analyze NOS1 localisation, activity, dimerisation, and its relationship with systolic function in ICM. The study has been carried out on left ventricular tissue obtained from explanted human hearts. Here we demonstrate that the upregulation of cardiac NOS1 is not accompanied by an increase in NOS activity, due in part to the alterations found in molecules involved in the regulation of its activity. We observed partial translocation of NOS1 to the sarcolemma in ischemic hearts, and a direct relationship between its protein levels and systolic ventricular function. Our findings indicate that NOS1 may be significant in the pathophysiology of human ischemic heart disease with a preservative role in maintaining myocardial homeostasis.

Background Nitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin ( BH 4)—a cofactor required for normal nitric oxide synthase function—supporting a pathophysiological link. Genetically augmenting BH 4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH 4 administration. We tested whether the primary cellular target of BH 4 is the cardiomyocyte or whether other novel mechanisms are invoked. Methods and Results Mice with cardiomyocyte‐specific overexpression of GTP cyclohydrolase 1 ( mGCH 1) and wild‐type littermates underwent transverse aortic constriction. The mGCH 1 mice had markedly increased myocardial BH 4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction–induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH 4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction. Conclusions BH 4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH 4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.

Atrial fibrillation (AF) is a growing public health burden, and its treatment remains a challenge. AF leads to electrical remodeling of the atria, which in turn promotes AF maintenance and resistance to treatment. Although remodeling has long been a therapeutic target in AF, its causes remain poorly understood. We show that atrial-specific up-regulation of microRNA-31 (miR-31) in goat and human AF depletes neuronal nitric oxide synthase (nNOS) by accelerating mRNA decay and alters nNOS subcellular localization by repressing dystrophin translation. By shortening action potential duration and abolishing rate-dependent adaptation of the action potential duration, miR-31 overexpression and/or disruption of nNOS signaling recapitulates features of AF-induced remodeling and significantly increases AF inducibility in mice in vivo. By contrast, silencing miR-31 in atrial myocytes from patients with AF restores dystrophin and nNOS and normalizes action potential duration and its rate dependency. These findings identify atrial-specific up-regulation of miR-31 in human AF as a key mechanism causing atrial dystrophin and nNOS depletion, which in turn contributes to the atrial phenotype begetting this arrhythmia. miR-31 may therefore represent a potential therapeutic target in AF.

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.

Rationale: Neuronal nitric oxide synthase (nNOS) expression and activity in atria are markedly reduced from patients and goats with atrial fibrillation (AF). Whether loss of nNOS contributes to AF-induced atrial electrical remodelling and its upstream mechanism remain unclear.Methodology: Whole-cell patch clamp was used to record action potentials (APs) and ion currents. N/n: number of patients or mice/ number of myocytes.Results: Inhibition of nNOS by S-methylthiocitrulline (SMTC, N/n: 12/45) induces a significant reduction in AP duration (APD) and APD rate-dependent adaptation in human right atrial myocytes with sinus rhythm (SR, N/n: 14/52). In mice, nNOS inhibition (N/n: 4/9) or nNOS-/- (N/n: 9/28) reduce APD50 and 90 by 53% and 35%, respectively.Ionic investigations show SMTC increases atrial IKur by 60%, Ito by 34% and IK1 by 27% with no change in ICa or IKr. Computer modelling shows mimicking SMTC-induced increases in IKur and IK1 successfully retrieves experimental phenotype. Furthermore blocking IKur abolishes the effect of nNOS inhibition on APD and APD rate-dependent adaptation (SMTC: N/n: 6/11 vs vehicle: N/n: 8/16). Additionally, In vivo atrial burst stimulation shows nNOS-/- mice exhibit 2 folds higher AF inducibility.In AF, upregulation of atrial specific miR-31 results in accelerating nNOS mRNA and protein decay. Inhibition of miR-31 recovers the SMTC suppressed APD90 (N/n: 3/12 in each group) and APD rate-dependent adaptation. These effects are NO-mediated as they are reversed by SMTC. Whereas in SR patients, increasing miR-31(N/n: 4/30) reduces nNOS, shortens APD and suppresses APD rate-dependent adaptation (N/n: 3/14).Conclusions: In human and mammalian atrial myocytes, a reduction in nNOS, mediating by upregulation of miR31, is an important factor retrieving key hallmarks of atrial electrical remodelling and contributing to the atrial phenotype begetting AF.