Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency.

BACKGROUND: To develop an accurate stable isotope dilution assay for simultaneous quantification of creatine metabolites ornithine, arginine, creatine, creatinine, and guanidinoacetate in very small blood sample volumes to study creatine metabolism in mice. METHODS: Liquid-chromatography (C18) tandem mass spectrometry with butylation was performed in positive ionization mode. Stable isotope dilution assay with external calibration was applied to three different specimen types, plasma, whole blood and dried blood spot (DBS). RESULTS: Analytical separation, sensitivity, accuracy, and linearity of the assay were adequate. The stable isotope dilution assay in plasma revealed no significant bias to gold standard methods for the respective analytes. Compared to plasma, we observed an overestimate of creatine and creatinine (2- to 5-fold and 1.2- to 2-fold, respectively) in whole-blood and DBS, and an underestimate of arginine (2.5-fold) in DBS. Validation of the assay in mouse models of creatine deficiency revealed plasma creatine metabolite pattern in good accordance with those observed in human GAMT and AGAT deficiency. Single dose intraperitoneal application of ornithine in wild-type mice lead to fast ornithine uptake (Tmax ≤ 10 min) and elimination (T1/2=24 min), and a decline of guanidinoacetate. CONCLUSION: The assay is fast and reliable to study creatine metabolism and pharmacokinetics in mouse models of creatine deficiency.

Disturbed energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by creatine intake.

Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP-phosphocreatine phosphoryl exchange reaction mediated by creatine kinase. Given that the consequences of Cr depletion are incompletely understood, we assessed the morphological, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of l-arginine:glycine amidinotransferase (AGAT(-/-)), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hindlimb muscle of AGAT(-/-) mice. Compared with wild-type, the inorganic phosphate/ß-ATP ratio was increased fourfold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F(1)F(0)-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT(-/-) mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT(-/-) muscle fibres. Ischaemia resulted in exacerbation of the decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT(-/-) mice can be switched between Cr deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific creatine kinase deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the phosphocreatine-creatine kinase system.

Lower force and impaired performance during high-intensity electrical stimulation in skeletal muscle of GAMT-deficient knockout mice.

Force characteristics of skeletal muscle of knockout mice lacking creatine (Cr) due to a deletion of guanidinoacetate methyltransferase (GAMT) were studied in situ. Medial gastrocnemius muscles of anesthetized GAMT-deficient (GAMT-/-) and control (Con) littermates were stimulated at optimum length via the sciatic nerve at different stimulation frequencies (60-250 Hz). GAMT-/- mice showed reduced maximal tetanic and twitch force, reduced relative force at 60 Hz, and increased relaxation times. High-intensity fatigue protocols consisting of 30 successive isometric or dynamic contractions showed a strong reduction in force at the beginning of the series in GAMT-/- mice, followed by a smaller reduction compared with Con littermates toward the end of the series. Cr supplementation for 2 days in GAMT-/- animals (GAMT(Cr)-/-) resulted in normalization to Con values for relaxation times, relative force at lower stimulation frequencies, and relative force during 30 isometric contractions. Force per muscle mass, however, remained decreased. Furthermore,GAMT(Cr)-/- mice showed differences compared with both Con and unsupplemented animals in maximal rates of force rise and relaxation times during the isometric protocol as well as in force during the dynamic protocol. Our results show that the absence of Cr plays a direct role in relaxation times, maximal rate of force rise, and force production during high-intensity fatigue protocols. The lower force per muscle mass, however, is probably caused by other factors; i.e., high intracellular guanidinoacetate concentrations.

Effective long-term control of cardiac events with beta-blockers in a family with a common LQT1 mutation.

The congenital long QT syndrome (LQTS) is characterized by a prolonged QT interval on the surface electrocardiogram and an increased risk of recurrent syncope and sudden cardiac death. Mutations in seven genes have been identified as the molecular basis of LQTS. beta-blockers are the treatment of choice to reduce cardiac symptoms. However, long-term follow-up of genotyped families with LQTS has been rarely reported. We have clinically followed a four-generation family with LQTS being treated with beta-blocker therapy over a period of 23 years. Seven family members were carriers of two amino acid alterations in cis (V254M-V417M) in the cardiac potassium channel gene KCNQ1. Voltage-clamp recordings of mutant KCNQ1 protein in Xenopus oocytes showed that only the V254M mutation reduced the IKs current and that the effect of the V417M variant was negligible. The family exhibited the complete clinical spectrum of the disease, from asymptomatic patients to victims of sudden death before beta-blocker therapy. There was no significant reduction in QTc (556 +/- 40 ms(1/2) before therapy, 494 +/- 20 ms(1/2) during 17 years of treatment; n = 5 individuals). Of nine family members, one female died suddenly before treatment, three females of the second generation were asymptomatic, and four individuals of the third and fourth generation were symptomatic. All mutation carriers were treated with beta-blockers and remained asymptomatic for a follow-up up to 23 years. Long-term follow-up of a LQT1 family with a common mutation (V254M) being on beta-blocker therapy was effective and safe. This study underscores the importance of long-term follow-up in families with specific LQT mutations to provide valuable information for clinicians for an appropriate antiarrhythmic treatment.

Local anaesthetic sensitivities of cloned HERG channels from human heart: comparison with HERG/MiRP1 and HERG/MiRP1 T8A.

BACKGROUND: Myocardial potassium channels are complexes formed by different subunits. The subunit composition may influence the cardiotoxic action of local anaesthetics. The effects of amide local anaesthetics on HERG channels co-expressed with the putative subunit MiRP1 have not been established. It is also unclear if the common polymorphism MiRP1(T8A) that predisposes individuals to drug-induced cardiac arrhythmia increases local-anaesthetic sensitivity of HERG/MiRP1 channels. This may suggest the presence of genetic risk factors for local-anaesthetic-induced cardiac arrhythmia. METHODS: Whole-cell patch-clamp recordings and site-directed mutagenesis were combined to compare local anaesthetic sensitivities of cloned and mutated human potassium channel subunits. The ion channels were activated by a protocol that approximated ventricular action potentials. RESULTS: The amide local anaesthetics bupivacaine, levobupivacaine and ropivacaine inhibited HERG channels at toxicologically relevant concentrations, with IC(50) values of 20 (SEM 2) micro M (n=29), 10 (1) micro M (n=40) and 20 (2) micro M (n=49), respectively. Hill coefficients were close to unity. There were no indications of qualitative differences in channel inhibition between the three anaesthetics. The putative subunit MiRP1 did not alter local anaesthetic sensitivity of HERG channels. The common single nucleotide polymorphism producing MiRP1(T8A) did not increase local anaesthetic sensitivity of HERG/MiRP1 channels. CONCLUSIONS: Amide local anaesthetics target HERG and HERG/MiRP1 channels with identical potency. The effects on these ion currents may significantly contribute to local-anaesthetic-induced cardiac arrhythmia. MiRP1(T8A) does not seem to confer an increased risk of severe cardiac side-effects to carriers of this common polymorphism.

Biophysical properties of Kv3.1 channels in SH-SY5Y human neuroblastoma cells.

Biophysical properties of delayed rectifier K channels in the human neuroblastoma SH-SY5Y were established using patch clamp recordings. The whole cell K+ conductance activated at membrane potentials positive to -20 mV. The midpoint of current activation was 9.6 +/- 5.1 mV, the equivalent charge was 3.7 +/-.6. Whole-cell currents inactivated slightly with time constants of 700 ms and 5 s. The K+ currents were sensitive to micromolar concentrations of TEA and 4-aminopyridine. RT-PCR experiments amplified a cDNA fragment specific for human Kv3.1 channels. Activation gating parameters in outside-out patches were shifted by approximately 14 mV in the hyperpolarizing direction.

A novel long-QT 5 gene mutation in the C-terminus (V109I) is associated with a mild phenotype.

Mutations in the human minK gene KCNE1 have been linked to autosomal dominant and autosomal recessive long-QT (LQT) syndrome, a cardiac condition predisposing to ventricular arrhythmias. minK and KvLQT1, the LQT1 gene product, form a native cardiac K+ channel that regulates the slowly delayed rectifier potassium current I(Ks). We used single-strand conformation polymorphism and sequencing techniques to identify novel KCNE1 mutations in patients with a congenital LQT syndrome of unknown genetic origin. In 150 unrelated index patients a missense mutation (V109I) was identified that significantly reduced the wild-type I(Ks) current amplitude (by 36%) when coexpressed with KvLQT1 in Xenopus oocytes. Other biophysical properties of the I(Ks) channel were not altered. Since we observed incomplete penetrance (only one of two mutation carriers could be diagnosed by clinical criteria), and the family's history was unremarkable for sudden cardiac death, the 109I allele most likely causes a mild phenotype. This finding may have implications for the occurrence of "acquired" conditions for ventricular arrhythmias and thereby the potential cardiac risk for asymptomatic mutation carriers still remains to be determined.

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents I(TO) and I(SA).

The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (I(TO)) and neurons (I(SA)). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23-p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4. 1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1-specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C-terminal splice KCND3L variant with an insertion of 19 amino acids.

Characterization of human Kv4.2 mediating a rapidly-inactivating transient voltage-sensitive K+ current.

A human cDNA for the voltage-sensitive potassium channel subunit Kv4.2 has been cloned and functionally characterized. The human Kv4.2 (KCND2) gene was mapped at 7q31-32. Kv4.2 mRNA is prominently expressed in human brain. Relatively high concentrations of Kv4.2 mRNA occurred in mRNA preparations of amygdala, caudate nucleus, cerebellum, hippocampus, substantia nigra, and thalamus. Kv4.2 mRNA was not detected in human heart, kidney, liver, lung, pancreas, and skeletal muscle. The derived Kv4.2 open reading frame consists of 630 amino acids. In comparison to rat Kv4.2, the human Kv4.2 sequence is highly conserved showing amino acid sequence differences at five positions only. The Kv4.2 subunits were expressed heterologously in human embryonic kidney (293) cells and mediated a rapidly inactivating, A-type outward K+ current. The gating kinetics of the Kv4.2-mediated currents were very similar to those of rat Kv4.2-mediated currents. Both the Kv4.2 and Kv4.3 subunits have been implicated in mediating the transient outward K+ current Ito in rodent cardiac myocytes. In contrast we did not detect Kv4.2. but solely Kv4.3 mRNA in human heart RNA preparations. This may suggest that Kv4.2 subunits do not contribute to the rapid transient outward K+ current of atrial and ventricular myocytes in humans.

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

Guanidinoacetate methyltransferase deficiency: the first inborn error of creatine metabolism in man.

In two children with an accumulation of guanidinoacetate in brain and a deficiency of creatine in blood, a severe deficiency of guanidinoacetate methyltransferase (GAMT) activity was detected in the liver. Two mutant GAMT alleles were identified that carried a single base substitution within a 5' splice site or a 13-nt insertion and gave rise to four mutant transcripts. Three of the transcripts encode truncated polypeptides that lack a residue known to be critical for catalytic activity of GAMT. Deficiency of GAMT is the first inborn error of creatine metabolism. It causes a severe developmental delay and extrapyramidal symptoms in early infancy and is treatable by oral substitution with creatine.

Cloning and sequence analysis of human guanidinoacetate N-methyltransferase cDNA.

In a child with severe muscular hypotonia and extrapyramidal movement disorder, elevation of guanidinoacetate and deficiency of creatine in brain suggested a defect in biosynthesis of creatine at the level of guanidinoacetate methyltransferase (GAMT) (Stöckler, S. et al. (1994) Pediatr. Res. 36, 409-413). As a first step in the molecular analysis of GAMT deficiency, the cDNA for human GAMT was cloned using rat GAMT cDNA as a probe. For the coding region there is a 82.5% homology of the nucleotide sequence and 86.9% homology of the deduced amino acid sequence between human and rat.

Juvenile form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). A C-terminal extension causes instability but increases catalytic efficiency of arylsulfatase B.

A deficiency of the enzyme arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type arylsulfatase B.

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase B gene alleles causing variable disease phenotypes.

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase B (ASB), also known as N-acetylgalactosamine-4-sulfatase. Multiple clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of the human ASB gene facilitated the analysis of molecular defects underlying the different phenotypes. Conditions for PCR amplification of the entire open reading frame from genomic DNA and for subsequent direct automated DNA sequencing of the resulting DNA fragments were established. Besides two polymorphisms described elsewhere that cause methionine-for-valine substitutions in the arylsulfatase B gene, six new mutations in six patients were detected: four point mutations resulting in amino acid substitutions, a 1-bp deletion, and a 1-bp insertion. The point mutations were two G-to-A and two T-to-C transitions. The G-to-A transitions cause an arginine-for-glycine substitution at residue 144 in a homoallelic patient with a severe disease phenotype and a tyrosine-for-cysteine substitution at residue 521 in a potentially heteroallelic patient with the severe form of the disease. The T-to-C transitions cause an arginine-for-cysteine substitution at amino acid residue 192 in a homoallelic patient with mild symptoms and a proline-for-leucine substitution at amino acid 321 in a homoallelic patient with the intermediate form. The insertion between nucleotides T1284 and G1285 resulted in a loss of the 100 C-terminal amino acids of the wild-type protein and in the deletion of nucleotide C1577 in a 39-amino-acid C-terminal extension of the ASB polypeptide. Both mutations were detected in homoallelic patients with the severe form of the disease. Expression of mutant cDNAs encoding the four amino acid substitutions and the deletion resulted in severe reduction of both ASB protein levels and arylsulfatase enzyme activity in comparison with a wild-type control. The six mutations described in the present study were unique among 25 unrelated mucopolysaccharidosis VI patients, suggesting a broad molecular heterogeneity of the Maroteaux-Lamy syndrome.

Four novel mutant alleles of the arylsulfatase B gene in two patients with intermediate form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome).

Mucopolysaccharidosis type VI (MPSVI, Maroteaux-Lamy syndrome) is a lysosomal storage disease for which multiple clinical phenotypes have been described. A deficiency of the enzyme arylsulfatase B (ASB, N-acetylgalactosamine-4-sulfatase) is the cause of this autosomal recessively inherited disorder. The genotypes of two patients with an intermediate form of MPSVI have been determined by polymerase chain reaction (PCR) amplification of the entire open reading frame of the ASB gene and subsequent direct sequencing of both strands of the PCR fragments by an automated nonradioactive approach. In patient A, a C to T transition in allele I resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, exon 2), and a G to A transition in allele II leading to a Gln to Arg160 substitution (R160Q, exon 2) were detected. Patient B exhibited a 7-bp deletion in exon 1 of allele I resulting in a frame shift and a premature stop codon 33 triplets 3' of the site of deletion (delta G237-C243), and a C to T transition in allele II giving rise to a Trp to Arg152 substitution (R152W, exon 2). None of these four mutant alleles was present among 60 alleles of the ASB gene in unrelated controls, indicating that the former are not polymorphisms. These results emphasize the broad molecular heterogeneity of Maroteaux-Lamy syndrome and contribute to the establishment of a genotype/phenotype correlation in this disease.