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.

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.

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.

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.

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.

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.

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.

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.

Cardiac Energetics in Patients With Aortic Stenosis and Preserved Versus Reduced Ejection Fraction.

BACKGROUND: Why some but not all patients with severe aortic stenosis (SevAS) develop otherwise unexplained reduced systolic function is unclear. We investigate the hypothesis that reduced creatine kinase (CK) capacity and flux is associated with this transition. METHODS: We recruited 102 participants to 5 groups: moderate aortic stenosis (ModAS) (n=13), SevAS, left ventricular (LV) ejection fraction ≥55% (SevAS-preserved ejection fraction, n=37), SevAS, LV ejection fraction <55% (SevAS-reduced ejection fraction, n=15), healthy volunteers with nonhypertrophied hearts with normal systolic function (normal healthy volunteer, n=30), and patients with nonhypertrophied, non-pressure-loaded hearts with normal systolic function undergoing cardiac surgery and donating LV biopsy (non-pressure-loaded heart biopsy, n=7). All underwent cardiac magnetic resonance imaging and 31P magnetic resonance spectroscopy for myocardial energetics. LV biopsies (AS and non-pressure-loaded heart biopsy) were analyzed for CK total activity, CK isoforms, citrate synthase activity, and total creatine. Mitochondria-sarcomere diffusion distances were calculated by using serial block-face scanning electron microscopy. RESULTS: In the absence of failure, CK flux was lower in the presence of AS (by 32%, P=0.04), driven primarily by reduction in phosphocreatine/ATP (by 17%, P<0.001), with CK kf unchanged (P=0.46). Although lowest in the SevAS-reduced ejection fraction group, CK flux was not different from the SevAS-preserved ejection fraction group (P>0.99). Accompanying the fall in CK flux, total CK and citrate synthase activities and the absolute activities of mitochondrial-type CK and CK-MM isoforms were also lower (P<0.02, all analyses). Median mitochondria-sarcomere diffusion distances correlated well with CK total activity (r=0.86, P=0.003). CONCLUSIONS: Total CK capacity is reduced in SevAS, with median values lowest in those with systolic failure, consistent with reduced energy supply reserve. Despite this, in vivo magnetic resonance spectroscopy measures of resting CK flux suggest that ATP delivery is reduced earlier, at the moderate AS stage, where LV function remains preserved. These findings show that significant energetic impairment is already established in moderate AS and suggest that a fall in CK flux is not by itself a necessary cause of transition to systolic failure. However, because ATP demands increase with AS severity, this could increase susceptibility to systolic failure. As such, targeting CK capacity and flux may be a therapeutic strategy to prevent and treat systolic failure in AS.

Overexpression of mitochondrial creatine kinase preserves cardiac energetics without ameliorating murine chronic heart failure.

Mitochondrial creatine kinase (Mt-CK) is a major determinant of cardiac energetic status and is down-regulated in chronic heart failure, which may contribute to disease progression. We hypothesised that cardiomyocyte-specific overexpression of Mt-CK would mitigate against these changes and thereby preserve cardiac function. Male Mt-CK overexpressing mice (OE) and WT littermates were subjected to transverse aortic constriction (TAC) or sham surgery and assessed by echocardiography at 0, 3 and 6 weeks alongside a final LV haemodynamic assessment. Regardless of genotype, TAC mice developed progressive LV hypertrophy, dilatation and contractile dysfunction commensurate with pressure overload-induced chronic heart failure. There was a trend for improved survival in OE-TAC mice (90% vs 73%, P = 0.08), however, OE-TAC mice exhibited greater LV dilatation compared to WT and no functional parameters were significantly different under baseline conditions or during dobutamine stress test. CK activity was 37% higher in OE-sham versus WT-sham hearts and reduced in both TAC groups, but was maintained above normal values in the OE-TAC hearts. A separate cohort of mice received in vivo cardiac 31P-MRS to measure high-energy phosphates. There was no difference in the ratio of phosphocreatine-to-ATP in the sham mice, however, PCr/ATP was reduced in WT-TAC but preserved in OE-TAC (1.04 ± 0.10 vs 2.04 ± 0.22; P = 0.007). In conclusion, overexpression of Mt-CK activity prevented the changes in cardiac energetics that are considered hallmarks of a failing heart. This had a positive effect on early survival but was not associated with improved LV remodelling or function during the development of chronic heart failure.

Localized rest and stress human cardiac creatine kinase reaction kinetics at 3 T.

Changes in the kinetics of the creatine kinase (CK) shuttle are sensitive markers of cardiac energetics but are typically measured at rest and in the prone position. This study aims to measure CK kinetics during pharmacological stress at 3 T, with measurement in the supine position. A shorter "stressed saturation transfer" (StreST) extension to the triple repetition time saturation transfer (TRiST) method is proposed. We assess scanning in a supine position and validate the MR measurement against biopsy assay of CK activity. We report normal ranges of stress CK forward rate (kfCK ) for healthy volunteers and obese patients. TRiST measures kfCK in 40 min at 3 T. StreST extends the previously developed TRiST to also make a further kfCK measurement during <20 min of dobutamine stress. We test our TRiST implementation in skeletal muscle and myocardium in both prone and supine positions. We evaluate StreST in the myocardium of six healthy volunteers and 34 obese subjects. We validated MR-measured kfCK against biopsy assays of CK activity. TRiST kfCK values matched literature values in skeletal muscle (kfCK  = 0.25 ± 0.03 s-1 vs 0.27 ± 0.03 s-1 ) and myocardium when measured in the prone position (0.32 ± 0.15 s-1 ), but a significant difference was found for TRiST kfCK measured supine (0.24 ± 0.12 s-1 ). This difference was because of different respiratory- and cardiac-motion-induced B0 changes in the two positions. Using supine TRiST, cardiac kfCK values for normal-weight subjects were 0.15 ± 0.09 s-1 at rest and 0.17 ± 0.15 s-1 during stress. For obese subjects, kfCK was 0.16 ± 0.07 s-1 at rest and 0.17 ± 0.10 s-1 during stress. Rest myocardial kfCK and CK activity from LV biopsies of the same subjects correlated (R = 0.43, p = 0.03). We present an independent implementation of TRiST on the Siemens platform using a commercially available coil. Our extended StreST protocol enables cardiac kfCK to be measured during dobutamine-induced stress in the supine position.

Adaptation to HIF1α Deletion in Hypoxic Cancer Cells by Upregulation of GLUT14 and Creatine Metabolism.

Hypoxia-inducible factor 1α is a key regulator of the hypoxia response in normal and cancer tissues. It is well recognized to regulate glycolysis and is a target for therapy. However, how tumor cells adapt to grow in the absence of HIF1α is poorly understood and an important concept to understand for developing targeted therapies is the flexibility of the metabolic response to hypoxia via alternative pathways. We analyzed pathways that allow cells to survive hypoxic stress in the absence of HIF1α, using the HCT116 colon cancer cell line with deleted HIF1α versus control. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, and proteomic analysis and integrated the results. These showed surprisingly that in three-dimensional growth, a key regulatory step of glycolysis is Aldolase A rather than phosphofructokinase. Furthermore, glucose uptake could be maintained in hypoxia through upregulation of GLUT14, not previously recognized in this role. Finally, there was a marked adaptation and change of phosphocreatine energy pathways, which made the cells susceptible to inhibition of creatine metabolism in hypoxic conditions. Overall, our studies show a complex adaptation to hypoxia that can bypass HIF1α, but it is targetable and it provides new insight into the key metabolic pathways involved in cancer growth. IMPLICATIONS: Under hypoxia and HIF1 blockade, cancer cells adapt their energy metabolism via upregulation of the GLUT14 glucose transporter and creatine metabolism providing new avenues for drug targeting.

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

Aims: Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Methods and results: Mice were created over-expressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1 H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20 min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55 ± 4% in WT vs. 29 ± 4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Conclusions: Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

Impaired cardiac contractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine.

AIMS: Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. METHODS AND RESULTS: Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. CONCLUSIONS: Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.

Validation of diffusion tensor MRI measurements of cardiac microstructure with structure tensor synchrotron radiation imaging.

BACKGROUND: Diffusion tensor imaging (DTI) is widely used to assess tissue microstructure non-invasively. Cardiac DTI enables inference of cell and sheetlet orientations, which are altered under pathological conditions. However, DTI is affected by many factors, therefore robust validation is critical. Existing histological validation is intrinsically flawed, since it requires further tissue processing leading to sample distortion, is routinely limited in field-of-view and requires reconstruction of three-dimensional volumes from two-dimensional images. In contrast, synchrotron radiation imaging (SRI) data enables imaging of the heart in 3D without further preparation following DTI. The objective of the study was to validate DTI measurements based on structure tensor analysis of SRI data. METHODS: One isolated, fixed rat heart was imaged ex vivo with DTI and X-ray phase contrast SRI, and reconstructed at 100 µm and 3.6 µm isotropic resolution respectively. Structure tensors were determined from the SRI data and registered to the DTI data. RESULTS: Excellent agreement in helix angles (HA) and transverse angles (TA) was observed between the DTI and structure tensor synchrotron radiation imaging (STSRI) data, where HADTI-STSRI = -1.4° ± 23.2° and TADTI-STSRI = -1.4° ± 35.0° (mean ± 1.96 standard deviation across all voxels in the left ventricle). STSRI confirmed that the primary eigenvector of the diffusion tensor corresponds with the cardiomyocyte long-axis across the whole myocardium. CONCLUSIONS: We have used STSRI as a novel and high-resolution gold standard for the validation of DTI, allowing like-with-like comparison of three-dimensional tissue structures in the same intact heart free of distortion. This represents a critical step forward in independently verifying the structural basis and informing the interpretation of cardiac DTI data, thereby supporting the further development and adoption of DTI in structure-based electro-mechanical modelling and routine clinical applications.

Protective Effect of Creatine Elevation against Ischaemia Reperfusion Injury Is Retained in the Presence of Co-Morbidities and during Cardioplegia.

AIMS: Ischaemic heart disease is most prevalent in the ageing population and often exists with other comorbidities; however the majority of laboratory research uses young, healthy animal models. Several recent workshops and focus meetings have highlighted the importance of using clinically relevant models to help aid translation to realistic patient populations. We have previously shown that mice over-expressing the creatine transporter (CrT-OE) have elevated intracellular creatine levels and are protected against ischaemia-reperfusion injury. Here we test whether elevating intracellular creatine levels retains a cardioprotective effect in the presence of common comorbidities and whether it is additive to protection afforded by hypothermic cardioplegia. METHODS AND RESULTS: CrT-OE mice and wild-type controls were subjected to transverse aortic constriction for two weeks to induce compensated left ventricular hypertrophy (LVH). Hearts were retrogradely perfused in Langendorff mode for 15 minutes, followed by 20 minutes ischaemia and 30 minutes reperfusion. CrT-OE hearts exhibited significantly improved functional recovery (Rate pressure product) during reperfusion compared to WT littermates (76% of baseline vs. 59%, respectively, P = 0.02). Aged CrT-OE mouse hearts (78±5 weeks) also had enhanced recovery following 15 minutes ischaemia (104% of baseline vs. 67%, P = 0.0007). The cardioprotective effect of hypothermic high K+ cardioplegic arrest, as used during cardiac surgery and donor heart transplant, was further enhanced in prolonged ischaemia (90 minutes) in CrT-OE Langendorff perfused mouse hearts (76% of baseline vs. 55% of baseline as seen in WT hearts, P = 0.02). CONCLUSIONS: These observations in clinically relevant models further support the development of modulators of intracellular creatine content as a translatable strategy for cardiac protection against ischaemia-reperfusion injury.

Refinement of analgesia following thoracotomy and experimental myocardial infarction using the Mouse Grimace Scale.

NEW FINDINGS: What is the central question of this study? There is an ethical imperative to optimize analgesia protocols for laboratory animals, but this is impeded by our inability to recognize pain reliably. We examined whether the Mouse Grimace Scale (MGS) provides benefits over a standard welfare scoring system for identifying a low level of pain in the frequently used murine surgical model of myocardial infarction. What is the main finding and its importance? Low-level pain, responsive to analgesia, was detected by MGS but not standard methods. In this model, most of the pain is attributable to the thoracotomy, excepted in mice with very large infarcts. This approach represents a model for assessing postsurgical analgesia in rodents. The Mouse Grimace Scale (MGS) was developed for assessing pain severity, but the general applicability to complex postsurgical pain has not been established. We sought to determine whether the MGS provides benefits over and above a standard welfare scoring system for identifying pain in mice following experimental myocardial infarction. Female C57BL/6J mice (n = 60), anaesthetized with isoflurane, were subjected to thoracotomy with ligation of a coronary artery or sham procedure. A single s.c. dose of buprenorphine (1.1 mg kg(-1) ) was given at the time of surgery and pain assessed at 24 h by MGS and a procedure-specific welfare scoring system. In some animals, a second dose of 0.6 mg kg(-1) buprenorphine was given and pain assessment repeated after 30 min. The MGS was scored from multiple photographs by two independent blinded observers with good correlation (r = 0.98). Using the average MGS score of both observers, we identified a subset of mice with low scores that were not considered to be in pain by the welfare scoring system or by single observer MGS. These mice showed a significant improvement with additional analgesia, suggesting that this low-level pain is real. Pain attributable to the myocardial injury, as opposed to thoracotomy, persisted at 24 h only in mice with large infarcts >40%. In conclusion, the use of a multi-observer, post hoc version of the MGS is a sensitive tool to assess the efficacy of postsurgical analgesic protocols. Following surgical induction of myocardial infarction, we identified a significant proportion of mice that were in low-level pain at 24 h that were not identified by other assessment methods.

Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart.

BACKGROUND: Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury. OBJECTIVES: To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse. METHODS AND RESULTS: CrT-OE mice were pre-selected for creatine levels 20-100% above wild-type using in vivo (1)H-MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction. CONCLUSIONS: Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity.

Metabolic flux as a predictor of heart failure prognosis.

The creatine kinase (CK) system is thought to play an integral role in maintaining levels of chemical energy in the form of ATP, which is essential for normal cardiac function. In the failing heart, it has long been established that multiple components of CK energy metabolism are commonly impaired and that these correlate with disease severity. A recent study published in Science Translational Medicine adds significantly to this body of evidence by demonstrating that the rate of ATP transfer via CK, measured noninvasively by magnetic resonance spectroscopy, is an independent predictor of adverse clinical outcome in patients with nonischemic cardiomyopathy. This finding invites speculation on the future role of metabolic imaging for risk stratification in patients with heart failure. The authors further assert an implied causal role for energetics in disease progression. Although this is not supported by recent findings in loss-of-function mouse models, there is, nonetheless, a strong argument for the development of novel metabolic therapies for the failing heart.