Liam Sebag-Montefiore

A search for type 1 diabetes susceptibility genes in families from the United Kingdom

Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (λs = 2.5; Refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at λs = 3 and lod = –2, of which two showed evidence of linkage: chromosome 10p13–p11 (maximum lod score (MLS) = 4.7, P = 3 × 10 –6, λs = 1.56) and chromosome 16q22–16q24 (MLS = 3.4, P = 6.5 × 10–5, λ s = 1.6). These and other novel regions, including chromosome 14q12–q21 and chromosome 19p13–19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.

Reduced Inotropic Reserve and Increased Susceptibility to Cardiac Ischemia/Reperfusion Injury in Phosphocreatine-Deficient Guanidinoacetate-N-Methyltransferase–Knockout Mice

Background—The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase–knockout (GAMT−/−) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT−/− mice. Methods and Results—GAMT−/− mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and β-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT−/− mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT−/− hearts (101±8 mm Hg in WT versus 59±7 mm Hg in GAMT−/−; P<0.05). 31P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT−/− hearts utilized P-GA. During ischemia/reperfusion, GAMT−/− hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23±9 in WT and 51±5 mm Hg in GAMT−/− during reperfusion; P<0.05) and incomplete resynthesis of P-GA. Conclusions—GAMT−/− mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT−/− mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.

Supranormal Myocardial Creatine and Phosphocreatine Concentrations Lead to Cardiac Hypertrophy and Heart Failure Insights From Creatine Transporter–Overexpressing Transgenic Mice

Background— Heart failure is associated with deranged cardiac energy metabolism, including reductions of creatine and phosphocreatine. Interventions that increase myocardial high-energy phosphate stores have been proposed as a strategy for treatment of heart failure. Previously, it has not been possible to increase myocardial creatine and phosphocreatine concentrations to supranormal levels because they are subject to tight regulation by the sarcolemmal creatine transporter (CrT). Methods and Results— We therefore created 2 transgenic mouse lines overexpressing the myocardial creatine transporter (CrT-OE). Compared with wild-type (WT) littermate controls, total creatine (by high-performance liquid chromatography) was increased in CrT-OE hearts (66±6 nmol/mg protein in WT versus 133±52 nmol/mg protein in CrT-OE). Phosphocreatine levels (by 31P magnetic resonance spectroscopy) were also increased but to a lesser extent. Surprisingly, CrT-OE mice developed left ventricular (LV) dilatation (LV end-diastolic volume: 21.5±4.3 &mgr;L in WT versus 33.1±9.6 &mgr;L in CrT-OE; P=0.002), substantial LV dysfunction (ejection fraction: 64±9% in WT versus 49±13% in CrT-OE; range, 22% to 70%; P=0.003), and LV hypertrophy (by 3-dimensional echocardiography and magnetic resonance imaging). Myocardial creatine content correlated closely with LV end-diastolic volume (r=0.51, P=0.02), ejection fraction (r=−0.74, P=0.0002), LV weight (r=0.59, P=0.006), LV end-diastolic pressure (r=0.52, P=0.02), and dP/dtmax (r=−0.69, P=0.0008). Despite increased creatine and phosphocreatine levels, CrT-OE hearts showed energetic impairment, with increased free ADP concentrations and reduced free-energy change levels. Conclusions— Overexpression of the CrT leads to supranormal levels of myocardial creatine and phosphocreatine, but the heart is incapable of keeping the augmented creatine pool adequately phosphorylated, resulting in increased free ADP levels, LV hypertrophy, and dysfunction. Our data demonstrate that a disturbance of the CrT-mediated tight regulation of cardiac energy metabolism has deleterious functional consequences. These findings caution against the uncritical use of creatine as a therapeutic agent in heart disease.

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. # Novelty and Significance {#article-title-47}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.

In vivo cardiac 1H-MRS in the mouse

The mouse is the predominant animal model to study the effect of gene manipulations. Imaging techniques to define functional effects on the heart caused by genomic alterations are becoming increasingly routine in mice, yet methods for in vivo investigation of metabolic phenotypes in the mouse heart are lacking. In this work, cardiac 1H‐MRS was developed and applied in mouse hearts in vivo using a single‐voxel technique (PRESS). In normal C57Bl/6J mice, stability and reproducibility achieved by dedicated cardiac and respiratory gating was demonstrated by measuring amplitude and zero‐order phase changes of the unsuppressed water signal. Various cardiac metabolites, such as creatine, taurine, carnitine, or intramyocardial lipids were successfully detected and quantified relative to the total water content in voxels as small as 2 μl, positioned in the interventricular septum. The method was applied to a murine model of guanidinoacetate N‐methyltransferase (GAMT) deficiency, which is characterized by substantially decreased myocardial creatine levels. Creatine deficiency was confirmed noninvasively in myocardium of anesthetized GAMT‐/‐ mice. This is the first study to report the application of cardiac 1H‐MRS in mice in vivo. Magn Reson Med 52:1029–1035, 2004.

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.

Mice over-expressing the myocardial creatine transporter develop progressive heart failure and show decreased glycolytic capacity.

The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32- week-old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel (1)H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the hearts metabolic enzymes and identified seven proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: alpha- and beta-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59+/-0.02 micromol ATP produced/microg protein/min; CrT-OE, 0.31+/-0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102+/-3 micromol/g wet myocardium; CrT-OE, 78+/-13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the beta-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study demonstrates that chronically increased myocardial [Cr] in the CrT-OE model leads to the development of progressive hypertrophy and heart failure, which may be mediated by a compromise in glycolytic capacity at the level of enolase.

Abnormal cardiac morphology, function and energy metabolism in the dystrophic mdx mouse: an MRI and MRS study.

Patients with muscular dystrophy have abnormal cardiac function and decreased high-energy phosphate metabolism. Here, we have determined whether the 8 month old mdx mouse, an animal model of muscular dystrophy, also has abnormal cardiac function and energetics. In vivo cardiac MRI revealed 33% and 104% larger right ventricular end-diastolic and end-systolic volumes, respectively, and 17% lower right ventricular ejection fractions in mdx mice compared with controls. Evidence of left ventricular diastolic dysfunction included 18% lower peak filling rates in mdx mouse hearts. Abnormal cardiac function was accompanied by necrosis and lower citrate synthase activity in the mdx mouse heart, suggesting decreased mitochondrial content. Decreased mitochondrial numbers were associated with 38% lower phosphocreatine concentration, 22% lower total creatine, 36% higher cytosolic free ADP concentration and 1.3 kJ/mol lower free-energy available from ATP hydrolysis in whole isolated, perfused mdx mouse hearts than in controls. Transsarcolemmal creatine uptake was 12% lower in mdx mouse hearts. We conclude that the absence of dystrophin in adult mdx mouse heart, as in the heart of human patient, is associated with right ventricular dilatation, left ventricular diastolic dysfunction and abnormal energy metabolism.

Serial high resolution 3D–MRI after aortic banding in mice: band internalization is a source of variability in the hypertrophic response

AbstractTransverse aortic constriction (TAC) is used as a model of left ventricular hypertrophy and failure; however, there is extensive variability in the hypertrophic response. In 43 mice that underwent TAC with a 7–0 polypropylene suture, 13 were identified by echocardiography with initial LV hypertrophy that halted or regressed over time. Post–mortem examination on 7 of these mice found the constricting band to be intact, but partially internalized into the aortic lumen, allowing blood flow around the stenosis. To confirm this prospectively in vivo we then followed 12 mice after TAC for 6 weeks, using a new high resolution 3D–MRI method to measure minimal aortic arch cross–sectional area (CSA). Three of the 12 mice developed a significantly increased aortic CSA (0.31 ± 0.15 on day 2 vs. 1.11 ± 0.29 mm2 on day 42; P < 0.05), which was independently confirmed by dissection. These mice had internalized part of the band within the aortic lumen and, by week 6, showed significantly less LV hypertrophy and better systolic function. Nine of the 12 mice showed no change in aortic CSA. Band internalization could be prevented when two banding sutures were placed side–by–side (n = 10). This is the first observation that a significant subset of animals following TAC bypass the stenosis resulting in partial regression of hypertrophy.

Cardiac phenotype of mitochondrial creatine kinase knockout mice is modified on a pure C57BL/6 genetic background.

UNLABELLED Discrepant results for the phenotype of mitochondrial creatine kinase knockout mice (Mt-CK(-/-)) could be due to mixed genetic background and use of non-littermate controls. We therefore backcrossed with C57BL/6J for >8 generations, followed by extensive in vivo cardiac phenotyping. Echocardiography and in vivo LV haemodynamics were performed in independent cohorts at 20-40 weeks and 1 year. No significant differences were observed for ECG, LV volumes, pressures, and systolic or diastolic function compared to littermate controls. Furthermore, responses to dobutamine were not different, indicating preserved contractile reserve. Contrary to published reports using Mt-CK(-/-) on a mixed background, we observed normal LV weights even in year old mice, and gene expression of common hypertrophic markers were not elevated. However, previously undetected adaptations were observed: an increase in activity of the cytosolic MM-CK isoenzyme (+20% vs WT, P=0.0009), and of citrate synthase (+18% vs WT, P=0.0007), a marker for mitochondrial volume. In a 3-week voluntary wheel running protocol, Mt-CK(-/-) ran significantly less per day (P=0.009) and attained lower maximum speed compared to controls (P=0.0003), suggesting impaired skeletal muscle function. MM-CK isoenzyme activity was significantly elevated in soleus but not gastrocnemius muscle of KO mice, and citrate synthase activities were normal in both, suggesting compensatory mechanisms are incomplete in skeletal muscle. CONCLUSIONS in contrast to previous reports using a mixed genetic background, Mt-CK(-/-) on a C57BL/6 background do not develop LV hypertrophy or dysfunction even up to 1 year, and this may be explained by a compensatory increase in MM-CK activity and mitochondrial volume.