Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

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.

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.

Fifteen-year follow-up of Italian families affected by arginine glycine amidinotransferase deficiency.

BACKGROUND: Arginine:glycine amidinotransferase deficiency (AGAT-d) is a very rare inborn error of creatine synthesis mainly characterized by absence of brain Creatine (Cr) peak, intellectual disability, severe language impairment and behavioural disorder and susceptible to supplementary Cr treatment per os. Serial examinations by magnetic resonance spectroscopy are required to evaluate Cr recovery in brain during treatment of high doses of Cr per os, which have been proved beneficial and effective in treating main clinical symptoms. A long term study with detailed reports on clinical, neurochemical and neuropsychological outcomes of the first Italian patients affected by AGAT-d here reported can represent a landmark in management of this disorder thus enhancing medical knowledge and clinical practice. RESULTS: We have evaluated the long term effects of Cr supplementation management in four Italian patients affected by AGAT-d, correlating specific treatments with serial clinical, biochemical and magnetic resonance spectroscopy examinations as well as the neuropsychological outcome by standardized developmental scales. Consecutive MRS examinations have confirmed that Cr depletion in AGAT-d patients is reversible under Cr supplementation. Cr treatment is considered safe and well tolerated but side effects, including weight gain and kidney stones, have been reported. CONCLUSIONS: Early treatment prevents adverse developmental outcome, while patients diagnosed and treated at an older age showed partial but significant cognitive recovery with clear improvements in adaptive functioning.

Creatine deficiency syndrome. A treatable myopathy due to arginine-glycine amidinotransferase (AGAT) deficiency.

We report two sisters, aged 11 and 6years, with AGAT deficiency syndrome (OMIM 612718) which is the least common creatine deficiency syndrome. They were born full-term to consanguineous parents and had moderate developmental delay. Examination showed an important language delay, a progressive proximal muscular weakness in the lower limbs with Gowers sign and myopathic electromyography. Investigations revealed undetectable guanidinoacetate and low level of creatine in plasma and urine, characteristic findings of AGAT deficiency syndrome. Brain magnetic resonance spectroscopy showed a markedly reduced level of creatine. Guanidinoacetate methyltransferase (GATM) gene sequencing revealed a homozygous missense mutation in exon 4:c.608A>C, (p.Tyr203Ser). Thirteen months after beginning the treatment with oral creatine monohydrate 200mg/kg/day, then 400mg/kg/day, there was a dramatic improvement in muscle strength with Gowers sign disappearance in both patients, and a mild improvement in language and cognitive functions. AGAT deficiency syndrome should be considered in all patients with language retardation and cognitive impairment associated to a myopathy of unknown etiology such that early diagnosis must lead to creatine supplementation to cure the myopathy and improve language and cognitive function.

Developmental progress and creatine restoration upon long-term creatine supplementation of a patient with arginine:glycine amidinotransferase deficiency.

BACKGROUND: Arginine:glycineamidinotransferase (AGAT/GATM) deficiency has been described in 9 patients across 4 families. Here we describe the clinical outcome and response to creatine supplementation in a patient of the second family affected with AGAT deficiency-a 9-year-old girl. PATIENT AND METHODS: Delayed motor milestones were noticed from 4 months of age and at 14 months moderate hypotonia, developmental delay and failure to thrive. Laboratory studies revealed low plasma creatine as well as extremely low levels of guanidinoacetic acid in urine and plasma. Proton magnetic resonance spectroscopy (MRS) of the brain showed absence of creatine. DNA sequence analysis revealed a homozygous mutation (c.484+1G>T) in the AGAT/GATM gene. AGAT activity was not detectable in lymphoblasts and RNA analysis revealed a truncated mRNA (r.289_484del196) that is degraded via Nonsense Mediated Decay. At 16 months, Bayley's Infant Development Scale (BIDS) showed functioning at 43% of chronologic age. Oral creatine supplementation (up to 800 mg/kg/day) was begun. RESULTS: At age 9 years she demonstrated advanced academic performance. Partial recovery of cerebral creatine levels was demonstrated on MRS at 25 months of age. Brain MRS at 40 months of age revealed a creatine/NAA ratio of about 80% of that in age-matched controls. CONCLUSIONS: 8 years post initiation of oral creatine supplementation, patient demonstrates superior nonverbal and academic abilities, with average verbal skills. We emphasize that early diagnosis combined with early treatment onset of AGAT deficiency may lead to improvement of developmental outcome.

l-arginine:glycine amidinotransferase (AGAT) deficiency: clinical presentation and response to treatment in two patients with a novel mutation.

Creatine and creatine phosphate provide storage and transmission of phosphate-bound energy in muscle and brain. Of the three inborn errors of creatine metabolism causing brain creatine depletion, l-arginine:glycine amidinotransferase (AGAT) deficiency has been described in only two families. We describe clinical and biochemical features, magnetic resonance spectroscopy (MRS) findings and response to creatine supplementation in two siblings with a novel mutation in the AGAT-encoding GATM gene. The sister and brother were evaluated at age 12 and 18years, respectively, because of mild mental retardation, muscle weakness and low weight. Extensive work-up had previously yielded negative results. Electron microscopy of the muscle revealed tubular aggregates and the activity of respiratory chain complexes was decreased in the muscle. Urine organic acid concentrations normalized to urine creatinine concentration were all increased, suggesting a creatine metabolism disorder. Brain MRS was remarkable for absence of creatine. Urine guanidinoacetate levels by tandem mass spectrometry were low, suggesting AGAT deficiency. GATM sequencing revealed a homozygous single nucleotide insertion 1111_1112insA, producing a frame-shift at Met-371 and premature termination at codon 376. Eleven months after commencing treatment with oral creatine monohydrate 100mg/kg/day, repeat MRI/MRS showed significantly increased brain creatine in the sister and a slight increase in the older brother. The parents' impression of improved strength and stamina was substantiated by increased post-treatment versus pre-treatment scores in the Vineland Adaptive Behavior Scale, straight-arm raising and timed up-and-go tests. Similarly, there was an apparent improvement in cognitive function, with significantly increased IQ-scores in the sister and marginal improvement in the brother.

Creatine and creatine deficiency syndromes: biochemical and clinical aspects.

Creatine deficiency syndromes, which have only recently been described, represent a group of inborn errors of creatine synthesis (L-arginine-glycine amidinotransferase deficiency and guanidinoacetate methyltransferase deficiency) and transport (creatine transporter deficiency). Patients with creatine deficiency syndromes present with mental retardation expressive speech and language delay, and epilepsy. Patients with guanidinoacetate methyltransferase deficiency or creatine transporter deficiency may exhibit autistic behavior. The common denominator of these disorders is the depletion of the brain creatine pool, as demonstrated by in vivo proton magnetic resonance spectroscopy. For diagnosis, laboratory investigations start with analysis of guanidinoacetate, creatine, and creatinine in plasma and urine. Based on these findings, enzyme assays or DNA mutation analysis may be performed. The creatine deficiency syndromes are underdiagnosed, so the possibility should be considered in all children affected by unexplained mental retardation, seizures, and speech delay. Guanidinoacetate methyltransferase deficiency and arginine-glycine amidinotransferase deficiency are treatable by oral creatine supplementation, but patients with creatine transporter deficiency do not respond to this type of treatment.

Pre-symptomatic treatment of creatine biosynthesis defects.

Recent observations in two patients, one with AGAT deficiency (AGAT-D) and one with GAMT deficiency (GAMT-D), both diagnosed already at birth, provide first evidence for important therapeutic effects of pre-symptomatic treatment with creatine (Cr) supplementation in AGAT-D and Cr supplementation plus guanidinoacetate lowering strategies in GAMT-D. Although long-term data are lacking, the results suggest that complete prevention of neurological sequelae in early treated patients could be feasible (Battini et al., 2006; Schulze et al., 2006).

Arginine:glycine amidinotransferase (AGAT) deficiency in a newborn: early treatment can prevent phenotypic expression of the disease.

Arginine:glycine amidinotransferase deficiency is a treatable inborn error of creatine synthesis, characterized by mental retardation, language impairment, and behavioral disorders. We describe a patient in whom arginine:glycine amidinotransferase was diagnosed at birth and treated at 4 months with creatine supplementation. In contrast with his 2 older sisters, he had normal psychomotor development at 18 months.

Creatine depletion in a new case with AGAT deficiency: clinical and genetic study in a large pedigree.

Arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) deficiency is a recently recognized autosomal recessive inborn error of creatine biosynthesis, characterized by mental retardation and severe language impairment. We extensively investigated a third 5-year-old patient with AGAT deficiency, discovered in the pedigree of the same Italian family as the two index cases. At the age of 2 years he presented with psychomotor and language delay, and autistic-like behavior. Brain MRI was normal, but brain 1H-MRS disclosed brain creatine depletion, which almost completely normalized following creatine monohydrate supplementation. A remarkable clinical improvement paralleled the restoration of brain creatine concentration. AGAT and GAMT (guanidinoacetate:methyltransferase) genes were analyzed in the proband and in 26 relatives, including the two cousins with AGAT deficiency. Sequencing of the proband's AGAT gene disclosed the same homozygous mutation at nt position 9093 converting a tryptophan (TGG) to a stop codon (TAG) at residue 149 (W149X), as already described in the two previously reported cases. The proband's parents and 10 additional subjects of the pedigree were carriers for this mutation. AGAT deficiency was further confirmed by undetectable AGAT activity in the patient's lymphoblasts. Mutation analysis of the GAMT gene revealed a sequence variation in exon 6 (T209M), not in the proband, but in 15 additional subjects from the pedigree. The silent nature of this sequence variation is supported by its homozygosity in one AGAT deficient cousin and in one asymptomatic adult, both with normal GAMT activity.

Arginine:glycine amidinotransferase deficiency: the third inborn error of creatine metabolism in humans.

Arginine:glycine amidinotransferase (AGAT) catalyzes the first step of creatine synthesis, resulting in the formation of guanidinoacetate, which is a substrate for creatine formation. In two female siblings with mental retardation who had brain creatine deficiency that was reversible by means of oral creatine supplementation and had low urinary guanidinoacetate concentrations, AGAT deficiency was identified as a new genetic defect in creatine metabolism. A homozygous G-A transition at nucleotide position 9297, converting a tryptophan codon (TGG) to a stop codon (TAG) at residue 149 (T149X), resulted in undetectable cDNA, as investigated by reverse-transcription PCR, as well as in undetectable AGAT activity, as investigated radiochemically in cultivated skin fibroblasts and in virus-transformed lymphoblasts of the patients. The parents were heterozygous for the mutant allele, with intermediate residual AGAT activities. Recognition and treatment with oral creatine supplements may prevent neurological sequelae in affected patients.