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

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

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

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

SMN1 dosage analysis in spinal muscular atrophy from India

BACKGROUND: Spinal muscular atrophy (SMA) represents the second most common fatal autosomal recessive disorder after cystic fibrosis. Due to the high carrier frequency, the burden of this genetic disorder is very heavy in developing countries like India. As there is no cure or effective treatment, genetic counseling becomes very important in disease management. SMN1 dosage analysis results can be utilized for identifying carriers before offering prenatal diagnosis in the context of genetic counseling. METHODS: In the present study we analyzed the carrier status of parents and sibs of proven SMA patients. In addition, SMN1 copy number was determined in suspected SMA patients and parents of children with a clinical diagnosis of SMA. RESULTS: wenty nine DNA samples were analyzed by quantitative PCR to determine the number of SMN1 gene copies present, and 17 of these were found to have one SMN1 gene copy. The parents of confirmed SMA patients were found to be obligate carriers of the disease. Dosage analysis was useful in ruling out clinical suspicion of SMA in four patients. In a family with history of a deceased floppy infant and two abortions, both parents were found to be carriers of SMA and prenatal diagnosis could be offered in future pregnancies. CONCLUSION: SMN1 copy number analysis is an important parameter for identification of couples at risk for having a child affected with SMA and reduces unwarranted prenatal diagnosis for SMA. The dosage analysis is also useful for the counseling of clinically suspected SMA with a negative diagnostic SMA test

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