Synthesis and characterization of amine-functionalized graphene as a nitric oxide-generating coating for vascular stents.

Drug-eluting stents are commonly utilized for the treatment of coronary artery disease, where they maintain vessel patency and prevent restenosis. However, problems with prolonged vascular healing, late thrombosis, and neoatherosclerosis persist; these could potentially be addressed via the local generation of nitric oxide (NO) from endogenous substrates. Herein, we develop amine-functionalized graphene as a NO-generating coating on polylactic acid (PLA)-based bioresorbable stent materials. A novel catalyst was synthesized consisting of polyethyleneimine and polyethylene glycol bonded to graphene oxide (PEI-PEG@GO), with physicochemical characterization using x-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. In the presence of 10 µM S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP), PEI-PEG@GO catalyzed the generation of 62% and 91% of the available NO, respectively. Furthermore, PEI-PEG@GO enhanced and prolonged real-time NO generation from GSNO and SNAP under physiological conditions. The uniform coating of PEI-PEG@GO onto stent material is demonstrated via an optimized simple dip-coating method. The coated PLA maintains good biodegradability under accelerated degradation testing, while the PEI-PEG@GO coating remains largely intact. Finally, the stability of the coating was demonstrated at room temperature over 60 days. In conclusion, the innovative conjugation of polymeric amines with graphene can catalyze the generation of NO from S-nitrosothiols at physiologically relevant concentrations. This approach paves the way for the development of controlled NO-generating coatings on bioresorbable stents in order to improve outcomes in coronary artery disease.

Cardiac function and energetics in mice with combined genetic augmentation of creatine and creatine kinase activity.

Improving energy provision in the failing heart by augmenting the creatine kinase (CK) system is a desirable therapeutic target. However, over-expression of the creatine transporter (CrT-OE) has shown that very high creatine levels result in cardiac hypertrophy and dysfunction. We hypothesise this is due to insufficient endogenous CK activity to maintain thermodynamically favourable metabolite ratios. If correct, then double transgenic mice (dTg) overexpressing both CrT and the muscle isoform of CK (CKM-OE) would rescue the adverse phenotype. In Study 1, overexpressing lines were crossed and cardiac function assessed by invasive haemodynamics and echocardiography. This demonstrated that CKM-OE was safe, but too few hearts had creatine in the toxic range. In Study 2, a novel CrT-OE line was generated with higher, homogeneous, creatine levels and phenotyped as before. Myocardial creatine was 4-fold higher in CrT-OE and dTg hearts compared to wildtype and was associated with hypertrophy and contractile dysfunction. The inability of dTg hearts to rescue this phenotype was attributed to downregulation of CK activity, as occurs in the failing heart. Nevertheless, combining both studies in a linear regression analysis suggests a modest positive effect of CKM over a range of creatine concentrations. In conclusion, we confirm that moderate elevation of creatine is well tolerated, but very high levels are detrimental. Correlation analysis lends support to the theory that this may be a consequence of limited CK activity. Future studies should focus on preventing CKM downregulation to unlock the potential synergy of augmenting both creatine and CK in the heart.

Hyperpolarized 13C and 31P MRS detects differences in cardiac energetics, metabolism, and function in obesity, and responses following treatment.

Obesity is associated with important changes in cardiac energetics and function, and an increased risk of adverse cardiovascular outcomes. Multi-nuclear MRS and MRI techniques have the potential to provide a comprehensive non-invasive assessment of cardiac metabolic perturbation in obesity. A rat model of obesity was created by high-fat diet feeding. This model was characterized using in vivo hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate MRS, echocardiography and perfused heart 31P MRS. Two groups of obese rats were subsequently treated with either caloric restriction or the glucagon-like peptide-1 analogue/agonist liraglutide, prior to reassessment. The model recapitulated cardiovascular consequences of human obesity, including mild left ventricular hypertrophy, and diastolic, but not systolic, dysfunction. Hyperpolarized 13C and 31P MRS demonstrated that obesity was associated with reduced myocardial pyruvate dehydrogenase flux, altered cardiac tricarboxylic acid (TCA) cycle metabolism, and impaired myocardial energetic status (lower phosphocreatine to adenosine triphosphate ratio and impaired cardiac ΔG~ATP). Both caloric restriction and liraglutide treatment were associated with normalization of metabolic changes, alongside improvement in cardiac diastolic function. In this model of obesity, hyperpolarized 13C and 31P MRS demonstrated abnormalities in cardiac metabolism at multiple levels, including myocardial substrate selection, TCA cycle, and high-energy phosphorus metabolism. Metabolic changes were linked with impairment of diastolic function and were reversed in concert following either caloric restriction or liraglutide treatment. With hyperpolarized 13C and 31P techniques now available for human use, the findings support a role for multi-nuclear MRS in the development of new therapies for obesity.

Nitric Oxide Releasing Nanomaterials for Cardiovascular Applications.

A central paradigm of cardiovascular homeostasis is that impaired nitric oxide (NO) bioavailability results in a wide array of cardiovascular dysfunction including incompetent endothelium-dependent vasodilatation, thrombosis, vascular inflammation, and proliferation of the intima. Over the course of more than a century, NO donating formulations such as organic nitrates and nitrites have remained a cornerstone of treatment for patients with cardiovascular diseases. These donors primarily produce NO in the circulation and are not targeted to specific (sub)cellular sites of action. However, safe, and therapeutic levels of NO require delivery of the right amount to a precise location at the right time. To achieve these aims, several recent strategies aimed at therapeutically generating or releasing NO in living systems have shown that polymeric and inorganic (silica, gold) nanoparticles and nanoscale metal-organic frameworks could either generate NO endogenously by the catalytic decomposition of endogenous NO substrates or can store and release therapeutically relevant amounts of NO gas. NO-releasing nanomaterials have been developed for vascular implants (such as stents and grafts) to target atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, and cardiac tissue engineering. In this review, we discuss the advances in design and development of novel NO-releasing nanomaterials for cardiovascular therapeutics and critically examine the therapeutic potential of these nanoplatforms to modulate cellular metabolism, to regulate vascular tone, inhibit platelet aggregation, and limit proliferation of vascular smooth muscle with minimal toxic effects.

pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

Maintaining energy provision in the heart: the creatine kinase system in ischaemia-reperfusion injury and chronic heart failure.

The non-stop provision of chemical energy is of critical importance to normal cardiac function, requiring the rapid turnover of ATP to power both relaxation and contraction. Central to this is the creatine kinase (CK) phosphagen system, which buffers local ATP levels to optimise the energy available from ATP hydrolysis, to stimulate energy production via the mitochondria and to smooth out mismatches between energy supply and demand. In this review, we discuss the changes that occur in high-energy phosphate metabolism (i.e., in ATP and phosphocreatine) during ischaemia and reperfusion, which represents an acute crisis of energy provision. Evidence is presented from preclinical models that augmentation of the CK system can reduce ischaemia-reperfusion injury and improve functional recovery. Energetic impairment is also a hallmark of chronic heart failure, in particular, down-regulation of the CK system and loss of adenine nucleotides, which may contribute to pathophysiology by limiting ATP supply. Herein, we discuss the evidence for this hypothesis based on preclinical studies and in patients using magnetic resonance spectroscopy. We conclude that the correlative evidence linking impaired energetics to cardiac dysfunction is compelling; however, causal evidence from loss-of-function models remains equivocal. Nevertheless, proof-of-principle studies suggest that augmentation of CK activity is a therapeutic target to improve cardiac function and remodelling in the failing heart. Further work is necessary to translate these findings to the clinic, in particular, a better understanding of the mechanisms by which the CK system is regulated in disease.

S-nitrosocysteamine-functionalised porous graphene oxide nanosheets as nitric oxide delivery vehicles for cardiovascular applications.

Nitric oxide (NO) is a key signalling molecule released by vascular endothelial cells that is essential for vascular health. Low NO bioactivity is associated with cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure and NO donors are a mainstay of drug treatment. However, many NO donors are associated with the development of tolerance and adverse effects, so new formulations for controlled and targeted release of NO would be advantageous. Herein, we describe the design and characterisation of a novel NO delivery system via the reaction of acidified sodium nitrite with thiol groups that had been introduced by cysteamine conjugation to porous graphene oxide nanosheets, thereby generating S-nitrosated nanosheets. An NO electrode, ozone-based chemiluminescence and electron paramagnetic resonance spectroscopy were used to measure NO released from various graphene formulations, which was sustained at >5 × 10-10 mol cm-2 min-1 for at least 3 h, compared with healthy endothelium (cf. 0.5-4 × 10-10 mol cm-2 min-1). Single cell Raman micro-spectroscopy showed that vascular endothelial and smooth muscle cells (SMCs) took up graphene nanostructures, with intracellular NO release detected via a fluorescent NO-specific probe. Functionalised graphene had a dose-dependent effect to promote proliferation in endothelial cells and to inhibit growth in SMCs, which was associated with cGMP release indicating intracellular activation of canonical NO signalling. Chemiluminescence detected negligible production of toxic N-nitrosamines. Our findings demonstrate the utility of porous graphene oxide as a NO delivery vehicle to release physiologically relevant amounts of NO in vitro, thereby highlighting the potential of these formulations as a strategy for the treatment of cardiovascular diseases.

Mitochondria-targeted nanomedicines for cardiovascular applications.

Tweetable abstract Mitochondria are increasingly a target for drug delivery in cardiovascular diseases. This editorial describes how a nanomedicine approach may improve drug potency and efficacy in a safe and controlled manner.

Measuring cardiomyocyte cellular characteristics in cardiac hypertrophy using diffusion-weighted MRI.

PURPOSE: This paper presents a hierarchical modeling approach for estimating cardiomyocyte major and minor diameters and intracellular volume fraction (ICV) using diffusion-weighted MRI (DWI) data in ex vivo mouse hearts. METHODS: DWI data were acquired on two healthy controls and two hearts 3 weeks post transverse aortic constriction (TAC) using a bespoke diffusion scheme with multiple diffusion times ( Δ $$ \Delta $$ ), q-shells and diffusion encoding directions. Firstly, a bi-exponential tensor model was fitted separately at each diffusion time to disentangle the dependence on diffusion times from diffusion weightings, that is, b-values. The slow-diffusing component was attributed to the restricted diffusion inside cardiomyocytes. ICV was then extrapolated at Δ = 0 $$ \Delta =0 $$ using linear regression. Secondly, given the secondary and the tertiary diffusion eigenvalue measurements for the slow-diffusing component obtained at different diffusion times, major and minor diameters were estimated assuming a cylinder model with an elliptical cross-section (ECS). High-resolution three-dimensional synchrotron X-ray imaging (SRI) data from the same specimen was utilized to evaluate the biophysical parameters. RESULTS: Estimated parameters using DWI data were (control 1/control 2 vs. TAC 1/TAC 2): major diameter-17.4 µ $$ \mu $$ m/18.0 µ $$ \mu $$ m versus 19.2 µ $$ \mu $$ m/19.0 µ $$ \mu $$ m; minor diameter-10.2 µ $$ \mu $$ m/9.4 µ $$ \mu $$ m versus 12.8 µ $$ \mu $$ m/13.4 µ $$ \mu $$ m; and ICV-62%/62% versus 68%/47%. These findings were consistent with SRI measurements. CONCLUSION: The proposed method allowed for accurate estimation of biophysical parameters suggesting cardiomyocyte diameters as sensitive biomarkers of hypertrophy in the heart.

Synergistic effect on cardiac energetics by targeting the creatine kinase system: in vivo application of high-resolution 31P-CMRS in the mouse.

BACKGROUND: Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). METHODS AND RESULTS: Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57-mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. CONCLUSIONS: We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure.

Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure.

AIMS: Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. METHODS AND RESULTS: Study 1: homoarginine deficiency-female AGAT-/- and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT-/- had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6-7 weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency-as before, but AGAT-/- mice were given creatine-supplemented diet until 1 week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. CONCLUSIONS: Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial.

Graphene nanocomposites for real-time electrochemical sensing of nitric oxide in biological systems.

Nitric oxide (NO) signaling plays many pivotal roles impacting almost every organ function in mammalian physiology, most notably in cardiovascular homeostasis, inflammation, and neurological regulation. Consequently, the ability to make real-time and continuous measurements of NO is a prerequisite research tool to understand fundamental biology in health and disease. Despite considerable success in the electrochemical sensing of NO, challenges remain to optimize rapid and highly sensitive detection, without interference from other species, in both cultured cells and in vivo. Achieving these goals depends on the choice of electrode material and the electrode surface modification, with graphene nanostructures recently reported to enhance the electrocatalytic detection of NO. Due to its single-atom thickness, high specific surface area, and highest electron mobility, graphene holds promise for electrochemical sensing of NO with unprecedented sensitivity and specificity even at sub-nanomolar concentrations. The non-covalent functionalization of graphene through supermolecular interactions, including π-π stacking and electrostatic interaction, facilitates the successful immobilization of other high electrolytic materials and heme biomolecules on graphene while maintaining the structural integrity and morphology of graphene sheets. Such nanocomposites have been optimized for the highly sensitive and specific detection of NO under physiologically relevant conditions. In this review, we examine the building blocks of these graphene-based electrochemical sensors, including the conjugation of different electrolytic materials and biomolecules on graphene, and sensing mechanisms, by reflecting on the recent developments in materials and engineering for real-time detection of NO in biological systems.

Insights Into the Metabolic Aspects of Aortic Stenosis With the Use of Magnetic Resonance Imaging.

Pressure overload in aortic stenosis (AS) encompasses both structural and metabolic remodeling and increases the risk of decompensation into heart failure. A major component of metabolic derangement in AS is abnormal cardiac substrate use, with down-regulation of fatty acid oxidation, increased reliance on glucose metabolism, and subsequent myocardial lipid accumulation. These changes are associated with energetic and functional cardiac impairment in AS and can be assessed with the use of cardiac magnetic resonance spectroscopy (MRS). Proton MRS allows the assessment of myocardial triglyceride content and creatine concentration. Phosphorous MRS allows noninvasive in vivo quantification of the phosphocreatine-to-adenosine triphosphate ratio, a measure of cardiac energy status that is reduced in patients with severe AS. This review summarizes the changes to cardiac substrate and high-energy phosphorous metabolism and how they affect cardiac function in AS. The authors focus on the role of MRS to assess these metabolic changes, and potentially guide future (cellular) metabolic therapy in AS.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Influence of homoarginine on creatine accumulation and biosynthesis in the mouse.

Organisms obtain creatine from their diet or by de novo synthesis via AGAT (L-arginine:glycine amidinotransferase) and GAMT (Guanidinoacetate N-methyltrasferase) in kidney and liver, respectively. AGAT also synthesizes homoarginine (hArg), low levels of which predict poor outcomes in human cardiovascular disease, while supplementation maintains contractility in murine heart failure. However, the expression pattern of AGAT has not been systematically studied in mouse tissues and nothing is known about potential feedback interactions between creatine and hArg. Herein, we show that C57BL/6J mice express AGAT and GAMT in kidney and liver respectively, whereas pancreas was the only organ to express appreciable levels of both enzymes, but no detectable transmembrane creatine transporter (Slc6A8). In contrast, kidney, left ventricle (LV), skeletal muscle and brown adipose tissue must rely on creatine transporter for uptake, since biosynthetic enzymes are not expressed. The effects of creatine and hArg supplementation were then tested in wild-type and AGAT knockout mice. Homoarginine did not alter creatine accumulation in plasma, LV or kidney, whereas in pancreas from AGAT KO, the addition of hArg resulted in higher levels of tissue creatine than creatine-supplementation alone (P < 0.05). AGAT protein expression in kidney was downregulated by creatine supplementation (P < 0.05), consistent with previous reports of end-product repression. For the first time, we show that hArg supplementation causes a similar down-regulation of AGAT protein (P < 0.05). These effects on AGAT were absent in the pancreas, suggesting organ specific mechanisms of regulation. These findings highlight the potential for interactions between creatine and hArg that may have implications for the use of dietary supplements and other therapeutic interventions.

Assessing Myocardial Microstructure With Biophysical Models of Diffusion MRI.

Biophysical models are a promising means for interpreting diffusion weighted magnetic resonance imaging (DW-MRI) data, as they can provide estimates of physiologically relevant parameters of microstructure including cell size, volume fraction, or dispersion. However, their application in cardiac microstructure mapping (CMM) has been limited. This study proposes seven new two-compartment models with combination of restricted cylinder models and a diffusion tensor to represent intra- and extracellular spaces, respectively. Three extended versions of the cylinder model are studied here: cylinder with elliptical cross section (ECS), cylinder with Gamma distributed radii (GDR), and cylinder with Bingham distributed axes (BDA). The proposed models were applied to data in two fixed mouse hearts, acquired with multiple diffusion times, q-shells and diffusion encoding directions. The cylinderGDR-pancake model provided the best performance in terms of root mean squared error (RMSE) reducing it by 25% compared to diffusion tensor imaging (DTI). The cylinderBDA-pancake model represented anatomical findings closest as it also allows for modelling dispersion. High-resolution 3D synchrotron X-ray imaging (SRI) data from the same specimen was utilized to evaluate the biophysical models. A novel tensor-based registration method is proposed to align SRI structure tensors to the MR diffusion tensors. The consistency between SRI and DW-MRI parameters demonstrates the potential of compartment models in assessing physiologically relevant parameters.

The Pitfalls of in vivo Cardiac Physiology in Genetically Modified Mice - Lessons Learnt the Hard Way in the Creatine Kinase System.

In order to fully understand gene function, at some point, it is necessary to study the effects in an intact organism. The creation of the first knockout mouse in the late 1980's gave rise to a revolution in the field of integrative physiology that continues to this day. There are many complex choices when selecting a strategy for genetic modification, some of which will be touched on in this review, but the principal focus is to highlight the potential problems and pitfalls arising from the interpretation of in vivo cardiac phenotypes. As an exemplar, we will scrutinize the field of cardiac energetics and the attempts to understand the role of the creatine kinase (CK) energy buffering and transport system in the intact organism. This story highlights the confounding effects of genetic background, sex, and age, as well as the difficulties in interpreting knockout models in light of promiscuous proteins and metabolic redundancy. It will consider the dose-dependent effects and unintended consequences of transgene overexpression, and the need for experimental rigour in the context of in vivo phenotyping techniques. It is intended that this review will not only bring clarity to the field of cardiac energetics, but also aid the non-expert in evaluating and critically assessing data arising from in vivo genetic modification.

Subtle Role for Adenylate Kinase 1 in Maintaining Normal Basal Contractile Function and Metabolism in the Murine Heart.

AIMS: Adenylate kinase 1 (AK1) catalyses the reaction 2ADP ↔ ATP + AMP, extracting extra energy under metabolic stress and promoting energetic homeostasis. We hypothesised that increased AK1 activity would have negligible effects at rest, but protect against ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: Cardiac-specific AK1 overexpressing mice (AK1-OE) had 31% higher AK1 activity (P = 0.009), with unchanged total creatine kinase and citrate synthase activities. Male AK1-OE exhibited mild in vivo dysfunction at baseline with lower LV pressure, impaired relaxation, and contractile reserve. LV weight was 19% higher in AK1-OE males due to higher tissue water content in the absence of hypertrophy or fibrosis. AK1-OE hearts had significantly raised creatine, unaltered total adenine nucleotides, and 20% higher AMP levels (P = 0.05), but AMP-activated protein kinase was not activated (P = 0.85). 1H-NMR revealed significant differences in LV metabolite levels compared to wild-type, with aspartate, tyrosine, sphingomyelin, cholesterol all elevated, whereas taurine and triglycerides were significantly lower. Ex vivo global no-flow I/R, caused four-of-seven AK1-OE hearts to develop terminal arrhythmia (cf. zero WT), yet surviving AK1-OE hearts had improved functional recovery. However, AK1-OE did not influence infarct size in vivo and arrhythmias were only observed ex vivo, probably as an artefact of adenine nucleotide loss during cannulation. CONCLUSION: Modest elevation of AK1 may improve functional recovery following I/R, but has unexpected impact on LV weight, function and metabolite levels under basal resting conditions, suggesting a more nuanced role for AK1 underpinning myocardial energy homeostasis and not just as a response to stress.

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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.