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 mM (30 min), or the anti-neoplastic drug doxorubicin 15 mM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCd 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 wildtype 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

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

Dietary Supplementation with Homoarginine Preserves Cardiac Function in a Murine Model of Post-Myocardial Infarction Heart Failure

Low plasma homoarginine (HA) is an emerging biomarker for cardiovascular disease and an independent predictor of mortality in patients with heart failure. Plasma levels appear to reflect cardiac dysfunction, positively correlating with ejection fraction and inversely with circulating brain natriuretic peptide. However, whether this outcome is a bystander or cause-and-effect has yet to be established. Within the context of stroke, a direct causal relationship has been inferred because normal mice pretreated with 14 mg/L HA had a smaller stroke size. In the present study, we show for the first time that dietary supplementation with HA improves cardiac function in the setting of chronic heart failure, suggesting a novel preventive strategy and inferring that low HA levels may be inherently detrimental because of a loss of this effect

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

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction

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

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

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

Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65–85; 100–135; 160–250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC–MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00726-016-2236-x) contains supplementary material, which is available to authorized users

Functional recovery following hypothermic cardioplegic arrest.

<p>Isolated hearts were perfused for 15 min at baseline, then cardioplegic arrest for 90 min at 4°C (grey), and 30 min reperfusion, when CrT-OE hearts (n = 12) had improved functional recovery compared to WT hearts (n = 13). (a) rate pressure product, RPP; (b) left ventricular developed pressure, LVDP; (c) left ventricular systolic pressure, LVSP; (d) left ventricular end-diastolic pressure, LVEDP; (e) heart rate, HR. (f) Pearson's correlation analysis indicates a positive relationship between functional recovery and total creatine levels (WT hearts are black circles and CrT-OE white). Data shown as mean values ± SEM Comparisons between groups by two-way repeated measures (mixed model) ANOVA with a Bonferroni Post-hoc test. *P<0.05, **P<0.01</p