Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications.

Why is early-onset atrial fibrillation uncommon in patients with Duchenne muscular dystrophy? Insights from the mdx mouse.

Aims: A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated.

BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes.

Rationale: 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.

Objective: 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.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

Aims: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.

Atrial nitroso-redox balance and refractoriness following on-pump cardiac surgery: a randomized trial of atorvastatin.

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.

Correction: Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

[This corrects the article DOI: 10.1371/journal.pone.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac F MRS/MRI and temporal tracking of progenitor cells.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and F 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. FuGENE-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]).

In Vivo Tracking and H/F Magnetic Resonance Imaging of Biodegradable Polyhydroxyalkanoate/Polycaprolactone Blend Scaffolds Seeded with Labeled Cardiac Stem Cells.

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.

Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

Purpose: To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification.

Methods: Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T.

The Subcellular Localisation of Neuronal Nitric Oxide Synthase Determines the Downstream Effects of NO on Myocardial Function.

Aims: In healthy hearts, the neuronal nitric oxide synthase (nNOS) is predominantly localized to the sarcoplasmic reticulum (SR), where it regulates the ryanodine receptor Carelease channel (RyR2) and phospholamban (PLB) phosphorylation, and to a lesser extent to the sarcolemmal membrane where it inhibits the L-type Cacurrent (). However, in failing hearts, impaired relaxation and depressed inotropy are associated with a larger proportion of nNOS being localized to the sarcolemmal membrane. Whether there is a causal relationship between altered myocardial function and subcellular localization of nNOS remains to be assessed.

Protein Inhibitor of NOS1 Plays a Central Role in the Regulation of NOS1 Activity in Human Dilated Hearts.

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.

Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase.

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.

Human Ischemic Cardiomyopathy Shows Cardiac Nos1 Translocation and its Increased Levels are Related to Left Ventricular Performance.

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.

Tetrahydrobiopterin Protects Against Hypertrophic Heart Disease Independent of Myocardial Nitric Oxide Synthase Coupling.

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 (BH4)-a cofactor required for normal nitric oxide synthase function-supporting a pathophysiological link. Genetically augmenting BH4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH4 administration.

Evaluation of the role of miR-31-dependent reduction in dystrophin and nNOS on atrial-fibrillation-induced electrical remodelling in man.

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.

Adenoviral transduction of FRET-based biosensors for cAMP in primary adult mouse cardiomyocytes.

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.

Nitric oxide synthase regulation of cardiac excitation-contraction coupling in health and disease.

Significant advances in our understanding of the ability of nitric oxide synthases (NOS) to modulate cardiac function have provided key insights into the role NOS play in the regulation of excitation-contraction (EC) coupling in health and disease. Through both cGMP-dependent and cGMP-independent (e.g.

Regulation of endothelial nitric-oxide synthase (NOS) S-glutathionylation by neuronal NOS: evidence of a functional interaction between myocardial constitutive NOS isoforms.

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline.

Nitric oxide synthases in heart failure.

Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca(2+) homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology.

Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance.

Cardiomyocyte GTP cyclohydrolase 1 and tetrahydrobiopterin increase NOS1 activity and accelerate myocardial relaxation.

Rationale: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood.

Objective: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca2+ handling.

Nitric oxide-releasing agent, LA419, reduces atherogenesis in apolipoprotein E-deficient mice.

LA419 is a novel nitric oxide-donor with antioxidant properties. The effect of this compound on the development of atherosclerosis was investigated in apolipoprotein E-deficient mice. Male mice were randomized to receive vehicle or 5 mg/kg/day LA419 for 12 weeks.

Genetic background in apolipoprotein A-I and cystathionine beta-synthase deficiency.

Double heterozygous mice lacking one allele of Cbs and Apoa1 develop hyperhomocysteinemia and hypoalphalipoproteinemia together with moderate hypertension. To study the influence of the genetic background into this specific phenotype, four groups of male mice were established: control and double heterozygous groups in C57BL/6J and in C57BL/6J x 129 backgrounds, respectively. Nitric oxide levels, systolic blood pressure, plasma lipid parameters, arylesterase activity and aorta histology were analyzed as well as oligonucleotide array hybridization of liver RNA.