ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes.

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells.

Cellular homeostasis of creatine (CT), integral part of the energy buffering and transducing system connecting intracellular sites of ATP production and utilization, comprises of mechanisms that increase CT, i.e., biosynthesis and cellular uptake, and CT-lowering processes, such as export and non-enzymatic conversion to creatinine. The biosynthesis of CT is controlled by negative feedback loop via suppression of the rate-limiting enzyme arginine:glycine amidinotransferase (AGAT). Although the regulatory mechanism involved is not well understood, AGAT suppression is successfully used in patients with guanidinoacetate methyltransferase (GAMT) deficiency to reduce the neurotoxic accumulation of the AGAT-mediated guanidinoacetate production by supplementing patients with CT. Utilizing the CT-dependent feedback loop for the upregulation of AGAT expression may well represent a therapeutic target for an additional CT deficiency syndrome, the CT transporter (CrT) defect, for which no effective treatment option is available so far. We have used CRISPR to tag the C-terminus of AGAT with a nanoluc luciferase (NLuc) reporter in HAP1 cells. A biphasic decay of AGAT-NLuc in response to increasing extracellular CT was observed, whereas the decrease in AGAT-NLuc expression was directly proportional to the rise in intracellular CT levels with an approximate IC50 of 1-2 mM. CRISPR generated HAP1 CrT null cells and HAP1 CrT null cells stably expressing a CrT-GFP fusion protein further demonstrated that the biphasic response to extracellular CT is mediated by a high-affinity (Km 9-10 µM) CrT dependent, saturable mechanism and a CrT independent, unsaturable uptake process. The direct response to intracellular CT suggests the existence of an intracellular CT sensing system enabling a dynamic cell response to changing CT concentration that is relevant for cellular CT homeostasis.

Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids.

Arginine:glycine amidinotransferase (AGAT) catalyzes mainly two reactions that generate 1) L-homoarginine (hArg) from L-arginine and L-lysine (Kharg) and 2) guanidinoacetate (GAA) and L-ornithine from L-arginine and glycine (Kgaa). Previously, we found that pharmacological treatment of Becker muscular dystrophy (BMD) patients with metformin or L-citrulline resulted in antidromic effects on serum hArg and GAA concentrations, seemingly acting as an inhibitor and effector of AGAT activity, respectively. Here, we used data of this study as a model to determine Kharg and Kgaa values by using the concentrations of the participating amino acids measured in serum samples of the BMD patients. The study aimed to prove the general utility of this approach to investigate effects of amino acids and drugs on AGAT-catalyzed reactions in vivo in humans.

GATM-Mediated Creatine Biosynthesis Enables Maintenance of FLT3-ITD-Mutant Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), with the most common mutation being internal tandem duplications (ITD). The presence of FLT3-ITD in AML carries a particularly poor prognosis and renders therapeutic resistance. New druggable targets are thus needed in this disease. In this study, we demonstrate the effects of de novo creatine biosynthesis upregulation by FLT3-ITD on AML sustainability. Our data show that FLT3-ITD constitutively activates the STAT5 signaling pathway, which upregulates the expression of glycine amidinotransferase (GATM), the first rate-limiting enzyme of de novo creatine biosynthesis. Pharmacologic FLT3-ITD inhibition reduces intracellular creatinine levels through transcriptional downregulation of genes in the de novo creatine biosynthesis pathway. The same reduction can be achieved by cyclocreatine or genetic GATM knockdown with shRNA and is reflected in significant decrease of cell proliferation and moderate increase of cell apoptosis in FLT3-ITD-mutant cell lines. Those effects are at least partially mediated through the AMPK/mTOR signaling pathway. This study uncovers a previously uncharacterized role of creatine metabolic pathway in the maintenance of FLT3-ITD-mutant AML and suggests that targeting this pathway may serve as a promising therapeutic strategy for FLT3-ITD-positive AML. IMPLICATIONS: FLT3-ITD mutation in AML upregulates de novo creatine biosynthesis that we show can be suppressed to diminish the proliferation and survival of blast cells.

GATM and GAMT synthesize creatine locally throughout the mammalian body and within oligodendrocytes of the brain.

The enzymes glycine amidinotransferase, mitochondrial (GATM also known as AGAT) and guanidinoacetate N-methyltransferase (GAMT) function together to synthesize creatine from arginine, glycine, and S-Adenosyl methionine. Deficiency in either enzyme or the creatine transporter, CT1, results in a devastating neurological disorder, Cerebral Creatine Deficiency Syndrome (CCDS). To better understand the pathophysiology of CCDS, we mapped the distribution of GATM and GAMT at single cell resolution, leveraging RNA sequencing analysis combined with in vivo immunofluorescence (IF). Using the mouse as a model system, we find that GATM and GAMT are coexpressed in several tissues with distinct and overlapping cellular sources, implicating local synthesis as an important mechanism of creatine metabolism in numerous organs. Extending previous findings at the RNA level, our analysis demonstrates that oligodendrocytes express the highest level of Gatm and Gamt of any cell type in the body. We confirm this finding in the mouse brain by IF, where GATM localizes to the mitochondria of oligodendrocytes, whereas both oligodendrocytes and cerebral cortical neurons express GAMT. Interestingly, the latter is devoid of GATM. Single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) analysis of 4 brain regions highlights a similar primacy of oligodendrocytes in the expression of GATM and GAMT in the human central nervous system. Importantly, an active putative regulatory element within intron 2 of human GATM is detected in oligodendrocytes but not neurons.

Creatine-mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer.

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

Increased creatine demand during pregnancy in Arginine: Glycine Amidino-Transferase deficiency: a case report.

BACKGROUND: Creatine (Cr), an amino acid derivative, is one of the most important sources of energy acting as both a spatial and temporal energy buffer through its phosphorylated analogue phosphocreatine (PCr) and creatine kinase (CK). Maternal Cr biosynthesis and metabolism seem to play an important role in pregnancy, as shown in preclinical and in healthy human pregnancy studies. Patients with Arginine:Glycine Amidino-Transferase deficiency (AGAT-d), due to the deficit of the first enzyme involved in Cr synthesis, are at a disadvantage due to their failure to synthesize Cr and their dependence on external intake, in contrast to normal subjects, where changes in Cr biosynthesis supply their needs. We report the outcomes of a pregnancy in an AGAT-d woman, and the challenge we faced in managing her treatment with oral Cr to ensure optimal conditions for her fetus. CASE PRESENTATION: A 22-year-old AGAT-d woman referred to our Institute for the management of her first conception at 11 weeks of fetal gestational age. Sonographic monitoring at 20 w GA indicated a reduction of fetal growth, in particular of the head circumference that was below the 3rd centile. Biochemical monitoring of Cr in biological fluids of the mother revealed a decline of the Cr concentrations, in particular in the urine sample, requiring prompt correction of the Cr dose. At 35 weeks of gestation the patient delivered a male infant, heterozygous for GATM mutation, with normal brain Cr levels; at one year the baby achieved typical developmental milestones. CONCLUSIONS: This rare pregnancy demonstrates that Cr levels in the blood and urine of the mother with AGAT-d decreased since the first months of gestation. The increase of the Cr daily dose administered to the mother seems to have produced beneficial effects also on the fetus.

Reconstitution of the Ornithine Cycle with Arginine:Glycine Amidinotransferase to Engineer Escherichia coli into an Efficient Whole-Cell Catalyst of Guanidinoacetate.

Guanidino compounds can be synthesized by transamidination reactions using arginine as a guanidine group donor. The efficiency of guanidino biosynthesis is often affected by the supply of arginine and the inhibition of the coproduct ornithine. To alleviate this shortcoming, we designed a reconstituted ornithine cycle in Escherichia coli to engineer an efficient whole-cell catalyst for guanidinoacetate (GAA) production by introducing a heterogeneous arginine:glycine amidinotransferase (AGAT). To alleviate the inhibition of ornithine, a citrulline synthetic module was constructed and optimized by introducing a glutamine self-sufficient system. Then, to improve the pathway from citrulline to arginine, an aspartate self-sufficient system was introduced into the arginine synthetic module. By combining these modules (GAA, citrulline, and arginine synthetic modules), a reconstituted ornithine cycle was developed, which significantly improved the biocatalyst efficiency (3.9-fold increase). In the system, arginine was regenerated efficiently through the reconstituted ornithine cycle, which converted arginine from a substrate to a cofactor for the transamidination reaction, thereby relieving the ornithine inhibition. Moreover, the amidino group of GAA in this system was mainly supplied by carbon and nitrogen assimilation. After the engineering process, 8.61 g/L GAA (73.56 mM) with a productivity of 0.39 g/L/h was achieved in a 22 h bioconversion. To the best of our knowledge, this is the first time that GAA has been produced in E. coli. This reconstructed ornithine cycle could be used as a transamidination platform for amidino group supply and has potential applications in the biosynthesis of other guanidino compounds.

Analysis of L-arginine:glycine amidinotransferase-, creatine- and homoarginine-dependent gene regulation in the murine heart.

L-arginine:glycine amidinotransferase (AGAT) and its metabolites creatine and homoarginine (HA) have been linked to cardiovascular pathologies in both human and murine studies, but the underlying molecular mechanisms are poorly understood. Here, we report the first analysis of heart transcriptome variation using microarrays in an AGAT-deficient (AGAT-/-) mouse model to evaluate AGAT-, creatine- and HA-dependent gene regulation. Our data revealed significant differences of gene expression between AGAT-/- and wild-type (WT) mice, affecting cardiac energy metabolism (Fbp2, Ucp2), cardiac hypertrophy and fibrosis (Nppa, Ctgf), immune response (Fgl2), and the conduction system of the heart (Dsc2, Ehd4, Hcn2, Hcn4, Scn4a, Scn4b). All of these genes being expressed on WT level in creatine-supplemented mice. Using in silico analysis based on the GEO database we found that most of these candidate genes (Ctgf, Dsc2, Fbp2, Fgl2, Hcn2, Nppa)  revealed significant alterations in a WT mouse model of myocardial infarction underlining a pathophysiological relationship between AGAT metabolism and cardiovascular disease.

Homoarginine- and Creatine-Dependent Gene Regulation in Murine Brains with l-Arginine:Glycine Amidinotransferase Deficiency.

l-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.

Creatine and pregnancy outcomes, a prospective cohort study in low-risk pregnant women: study protocol.

INTRODUCTION: The creatine kinase circuit is central to the regulation of high-energy phosphate metabolism and the maintenance of cellular energy turnover. This circuit is fuelled by creatine, an amino acid derivative that can be obtained from a diet containing animal products, and by synthesis in the body de novo. A recent retrospective study conducted in a cohort of 287 pregnant women determined that maternal excreted levels of creatine may be associated with fetal growth. This prospective study aims to overcome some of the limitations associated with the previous study and thoroughly characterise creatine homeostasis throughout gestation in a low-risk pregnant population. METHODS AND ANALYSIS: This study is recruiting women with a singleton low-risk pregnancy who are attending Monash Health, in Melbourne, Australia. Maternal blood and urine samples, along with dietary surveys, are collected at five time points during pregnancy and then at delivery. Cord blood and placenta (including membranes and cord) are collected at birth. A biobank of tissue samples for future research is being established. Primary outcome measures will include creatine, creatine kinase and associated metabolites in antenatal bloods and urine, cord bloods and placenta, along with molecular analysis of the creatine transporter (SLC6A8) and synthesising enzymes L - arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) in placental tissues. Secondary outcome measures include dietary protein intake over pregnancy and any associations with maternal creatine, pregnancy events and birth outcomes. ETHICS AND DISSEMINATION: Ethical approval was granted in August 2015 from Monash Health (Ref: 14140B) and Monash University (Ref: 7785). Study outcomes will be disseminated at international conferences and published in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: ACTRN12618001558213; Pre-results.

Benefits and drawbacks of guanidinoacetic acid as a possible treatment to replenish cerebral creatine in AGAT deficiency.

Arginine-glycine amidinotransferase (AGAT) deficiency is a rare inherited metabolic disorder that severely affects brain bioenergetics. Characterized by mental retardation, language impairment, and behavioral disorders, AGAT deficiency is a treatable condition, where long-term creatine supplementation usually restores brain creatine levels and improves its clinical features. In some cases of AGAT deficiency, creatine treatment might be somewhat limited due to possible shortcomings in performance and transport of creatine to the brain. Guanidinoacetic acid (GAA), a direct metabolic precursor of creatine, has recently been suggested as a possible alternative to creatine to tackle brain creatine levels in experimental medicine. AGAT patients might benefit from oral GAA due to upgraded bioavailability and convenient utilization of the compound, while possible drawbacks (e.g. brain methylation issues, neurotoxicity, and hyperhomocysteinemia) should be accounted as well.

Effects of SLCO1B1 and GATM gene variants on rosuvastatin-induced myopathy are unrelated to high plasma exposure of rosuvastatin and its metabolites.

Myotoxicity is a significant factor contributing to the poor adherence and reduced effectiveness in the treatment of statins. Genetic variations and high drug plasma exposure are considered as critique causes for statin-induced myopathy (SIM). This study aims to explore the sequential influences of rosuvastatin (RST) pharmacokinetic and myopathy-related single-nucleotide polymorphisms (SNPs) on the plasma exposure to RST and its metabolites: rosuvastatin lactone (RSTL) and N-desmethyl rosuvastatin (DM-RST), and further on RST-induced myopathy. A total of 758 Chinese patients with coronary artery disease were enrolled and followed up SIM incidents for 2 years. The plasma concentrations of RST and its metabolites were determined through a validated ultra-performance liquid chromatography mass spectrometry method. Nine SNPs in six genes were genotyped by using the Sequenom MassArray iPlex platform. Results revealed that ABCG2 rs2231142 variations were highly associated with the plasma concentrations of RST, RSTL, and DM-RST (Padj < 0.01, FDR < 0.05). CYP2C9 rs1057910 significantly affected the DM-RST concentration (Padj < 0.01, FDR < 0.05). SLCO1B1 rs4149056 variant allele was significantly associated with high SIM risk (OR: 1.741, 95% CI: 1.180-2.568, P = 0.0052, FDR = 0.0468). Glycine amidinotransferase (GATM) rs9806699 was marginally associated with SIM incidents (OR: 0.617, 95% CI: 0.406-0.939, P = 0.0240, FDR = 0.0960). The plasma concentrations of RST and its metabolites were not significantly different between the SIM (n = 51) and control groups (n = 707) (all P > 0.05). In conclusion, SLCO1B1 and GATM genetic variants are potential biomarkers for predicting RST-induced myopathy, and their effects on SIM are unrelated to the high plasma exposure of RST and its metabolites.

Novel Biosynthesis, Metabolism and Physiological Functions of L-Homoarginine.

L-Homoarginine (hArg) ((2S)-amino-6-Carbamimidamidohexanoic acid) is a non-essential cationic amino acid that may be synthesised from the lysine catabolism or the transamination of its precursor (Arginine: Arg). These processes involve the use of the ornithine transcarbamoylase (OTC), an enzyme from the urea cycle or the arginine: glycine amidinotransferase (AGAT), an enzyme from the creatine biosynthesis pathway. These enzymes are tissue-specific, hence they synthesised L-hArg in animals and human organs such as the liver, kidneys, brains, and the small intestines. L-hArg plays some important roles in the pathophysiological conditions, endothelial functions, and the energy metabolic processes in different organs. These functions depend on the concentrations of the available LhArg in the body. These different concentrations of the L-hArg in the body are related to the different disease conditions such as the T2D mellitus, the cardiovascular and the cerebrovascular diseases, the chronic kidney diseases, the intrauterine growth restriction (IUGR) and the preeclampsia (PE) in pregnancy disorders, and even mortality. However, the applications of the L-hArg in both human and animal studies is in its juvenile stage, and the mechanism of action in this vital amino acid is not fully substantiated and requires more research attention. Hence, we review the evidence with the perspective of the LhArg usage in the monogastric and human nutrition and its related health implications.

Effects of single and combined metformin and L-citrulline supplementation on L-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications.

The L-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21-44 years): (1) supplementation with L-citrulline (3 × 5 g/d), the precursor of L-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of L-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of L-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. L-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and L-citrulline attenuated the effects obtained from their single administrations. Metformin, L-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or L-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and L-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of L-citrulline.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

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.

The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase.

Studies of virulence determinants in the bacterial phytopathogen Erwinia amylovora, the cause of devastating fire blight disease in apple and pear, have shown that HsvA, a putative amidinotransferase enzyme located in the Hrp pathogenicity island, is required for systemic infection in apple. However, the mechanism by which HsvA contributes to virulence is unclear. To investigate the role of HsvA in virulence, we carried out a series of biochemical and structural studies to characterize the amidinotransferase activity of HsvA. We found that HsvA displays a preference for linear aliphatic polyamines as the amidino acceptor substrate, especially for spermidine and putrescine (Km values of 33 µm and 3.9 mm, respectively). The three-dimensional structure, determined at 2.30 Å resolution using X-ray crystallography, revealed that the overall architecture of HsvA is similar to that of the human arginine-glycine amidinotransferase in the creatine biosynthesis pathway. The active site is located in the core of the protein at the base of a long, narrow substrate access channel. Specific amino acids near the entrance of the channel may serve as major determinants of the substrate specificity, including a glutamate residue at the rim of the channel entrance that appears to be positioned to interact with the distal primary amine in the putrescine substrate as well as the internal and distal amines in the spermidine substrate. These results suggest potential in vivo functions for HsvA as a virulence factor in fire blight and may also provide a basis for strategies to control fire blight by inhibiting HsvA activity.