Living without creatine: unchanged exercise capacity and response to chronic myocardial infarction in creatine-deficient mice.

RATIONALE: Creatine is thought to be involved in the spatial and temporal buffering of ATP in energetic organs such as heart and skeletal muscle. Creatine depletion affects force generation during maximal stimulation, while reduced levels of myocardial creatine are a hallmark of the failing heart, leading to the widely held view that creatine is important at high workloads and under conditions of pathological stress. OBJECTIVE: We therefore hypothesised that the consequences of creatine-deficiency in mice would be impaired running capacity, and exacerbation of heart failure following myocardial infarction. METHODS AND RESULTS: Surprisingly, mice with whole-body creatine deficiency due to knockout of the biosynthetic enzyme (guanidinoacetate N-methyltransferase [GAMT]) voluntarily ran just as fast and as far as controls (>10 km/night) and performed the same level of work when tested to exhaustion on a treadmill. Furthermore, survival following myocardial infarction was not altered, nor was subsequent left ventricular (LV) remodelling and development of chronic heart failure exacerbated, as measured by 3D-echocardiography and invasive hemodynamics. These findings could not be accounted for by compensatory adaptations, with no differences detected between WT and GAMT(-/-) proteomes. Alternative phosphotransfer mechanisms were explored; adenylate kinase activity was unaltered, and although GAMT(-/-) hearts accumulated the creatine precursor guanidinoacetate, this had negligible energy-transfer activity, while mitochondria retained near normal function. CONCLUSIONS: Creatine-deficient mice show unaltered maximal exercise capacity and response to chronic myocardial infarction, and no obvious metabolic adaptations. Our results question the paradigm that creatine is essential for high workload and chronic stress responses in heart and skeletal muscle.

A role for thioredoxin-interacting protein (Txnip) in cellular creatine homeostasis.

Creatine is important for energy metabolism, yet excitable cells such as cardiomyocytes do not synthesize creatine and rely on uptake via a specific membrane creatine transporter (CrT; SLC6A8). This process is tightly controlled with downregulation of CrT upon continued exposure to high creatine via mechanisms that are poorly understood. Our aim was to identify candidate endogenous CrT inhibitors. In 3T3 cells overexpressing the CrT, creatine uptake plateaued at 3 h in response to 5 mM creatine but peaked 33% higher (P < 0.01) in the presence of cycloheximide, suggesting CrT regulation depends on new protein synthesis. Global gene expression analysis identified thioredoxin-interacting protein (Txnip) as the only significantly upregulated gene (by 46%) under these conditions (P = 0.036), subsequently verified independently at mRNA and protein levels. There was no change in Txnip expression with exposure to 5 mM taurine, confirming a specific response to creatine rather than osmotic stress. Small-interfering RNA against Txnip prevented Txnip upregulation in response to high creatine, maintained normal levels of creatine uptake, and prevented downregulation of CrT mRNA. These findings were relevant to the in vivo heart since creatine-deficient mice showed 39.71% lower levels of Txnip mRNA, whereas mice overexpressing the CrT had 57.6% higher Txnip mRNA levels and 28.7% higher protein expression compared with wild types (mean myocardial creatine concentration 124 and 74 nmol/mg protein, respectively). In conclusion, we have identified Txnip as a novel negative regulator of creatine levels in vitro and in vivo, responsible for mediating substrate feedback inhibition and a potential target for modulating creatine homeostasis.

Myocardial infarction causes inflammation and leukocyte recruitment at remote sites in the myocardium and in the renal glomerulus.

RATIONALE AND OBJECTIVE: Acute myocardial infarction (AMI) results in the recruitment of leukocytes to injured myocardium. Additionally, myocardium remote to the infarct zone also becomes inflamed and is associated with adverse left ventricular remodelling. Renal ischaemic syndromes have been associated with remote organ inflammation and impaired function. Here, we tested the hypothesis that AMI results in remote organ (renal) inflammation. METHODS: Mice were subjected to either AMI, sham procedure or no procedure and the inflammatory response in peripheral blood, injured and remote myocardium, and kidneys was studied at 24 h. RESULTS: AMI resulted in increased circulating neutrophils (P < 0.001) and monocytes (P < 0.001). mRNA for inflammatory mediators significantly increased in infarcted myocardium and in remote myocardium. VCAM-1 mRNA was increased in both infarcted and remote myocardium. VCAM-1 protein was also increased in the kidneys of AMI mice (P < 0.05) and immunofluorescence revealed localisation of VCAM-1 to glomeruli, associated with leukocyte infiltration and increased local inflammatory mRNA expression. CONCLUSIONS: We conclude that in addition to local inflammation, AMI results in remote organ inflammation evidenced by (1) increased expression of mRNA for inflammatory cytokines, (2) marked upregulation of VCAM-1 in renal glomeruli, and (3) the recruitment and infiltration of leukocytes in the kidney.

Chronic creatine kinase deficiency eventually leads to congestive heart failure, but severity is dependent on genetic background, gender and age.

The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(-/-)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1 year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(-/-) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(-/-) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression.

Accelerating global left-ventricular function assessment in mice using reduced slice acquisition and three-dimensional guide-point modelling.

BACKGROUND: To investigate the utility of three-dimensional guide-point modeling (GPM) to reduce the time required for CMR evaluation of global cardiac function in mice, by reducing the number of image slices required for accurate quantification of left-ventricular (LV) mass and volumes. METHODS: Five female C57Bl/6 mice 8 weeks post myocardial infarction induced by permanent occlusion of the left coronary artery, and six male control (un-operated) C57Bl/6 mice, were subject to CMR examination under isoflurane anaesthesia. Contiguous short axis (SAX) slices (1 mm thick 7-9 slices) were obtained together with two long axis (LAX) slices in two chamber and four chamber orientations. Using a mathematical model of the heart to interpolate information between the available slices, GPM LV mass and volumes were determined using full slice (all SAX and two LAX), six slice (four SAX and two LAX) and four slice (two SAX and two LAX) analysis protocols. All results were compared with standard manual volumetric analysis using all SAX slices. RESULTS: Infarct size was 39.1±5.1% of LV myocardium. No significant differences were found in left ventricular mass and volumes between the standard and GPM full and six slice protocols in infarcted mice (113±10, 116±11, and 117±11 mg respectively for mass), or between the standard and GPM full, six and four slice protocols in control mice, (105±14, 106±10, 104±12, and 105±7 mg respectively for mass). Significant differences were found in LV mass (135±18 mg) and EF using the GPM four slice protocol in infarcted mice (p<0.05). CONCLUSION: GPM enables accurate analysis of LV function in mice with relatively large infarcts using a reduced six slice acquisition protocol, and in mice with normal/symmetrical left-ventricular topology using a four slice protocol.

Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart.

Cardiac ischemia-reperfusion experiments in the mouse are important in vivo models of human disease. Infarct size is a particularly important scientific readout as virtually all cardiocirculatory pathways are affected by it. Therefore, such measurements must be exact and valid. The histological analysis, however, remains technically challenging, and the resulting quality is often unsatisfactory. For this report we have scrutinized each step involved in standard double-staining histology. We have tested published approaches and challenged their practicality. As a result, we propose an improved and streamlined protocol, which consistently yields high-quality histology, thereby minimizing experimental noise and group sizes.

Ribose Supplementation Alone or with Elevated Creatine Does Not Preserve High Energy Nucleotides or Cardiac Function in the Failing Mouse Heart.

BACKGROUND: Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP) are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE). METHODS AND RESULTS: FOUR GROUPS WERE STUDIED: sham; myocardial infarction (MI); MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN) pool was decreased to a similar amount (8-14%) in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV) dysfunction (3-fold reduction in ejection fraction) and LV hypertrophy (32-47% increased mass). Ejection fraction closely correlated with infarct size independently of treatment (r(2) = 0.63, p<0.0001), but did not correlate with myocardial creatine or TAN levels. CONCLUSION: Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated.

Moderate elevation of intracellular creatine by targeting the creatine transporter protects mice from acute myocardial infarction.

AIMS: Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: CrT-OE mice were selected for left ventricular (LV) creatine 20-100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury. CONCLUSION: Elevation of myocardial creatine by 20-100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia.