Genotype-phenotype dilemma in a case of sudden cardiac death with the E1053K mutation and a deletion in the SCN5A gene.

Mutations in the cardiac sodium channel gene SCN5A may result in various arrhythmia syndromes such as long QT syndrome type 3 (LQTS), Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction diseases (CCD) and possibly dilated cardiomyopathy (DCM). In most of these inherited cardiac arrhythmia syndromes the phenotypical expression may range from asymptomatic phenotypes to sudden cardiac death (SCD). A 16-year-old female died during sleep. Autopsy did not reveal any explanation for her death and a genetic analysis was performed. A variant in the SCN5A gene (E1053K) that was previously described as disease causing was detected. Family members are carriers of the same E1053K variant, some even in a homozygous state, but surprisingly did not exhibit any pathological cardiac phenotype. Due to the lack of genotype-phenotype correlation further genetic studies were performed. A novel deletion in the promoter region of SCN5A was identified in the sudden death victim but was absent in other family members. These findings demonstrate the difficulties in interpreting the results of a family-based genetic screening and underline the phenotypic variability of SCN5A mutations.

[Magnetic resonance imaging of hypertrophic cardiomyopathy : evaluation of diastolic function]. / MRT-Bildgebung bei hypertropher Kardiomyopathie (HCM) : Evaluation der diastolischen Funktion.

Hypertrophic cardiomyopathy (HCM) has a prevalence of approximately 0.2% and is clinically asymptomatic in many patients or presents with unspecific symptoms. This explains the importance of imaging for the diagnosis of HCM as well as for the assessment of the clinical course. The definitive finding in HCM is myocardial hypertrophy with thickening of the ventricular wall ≥ 15 mm. While echocardiography is an excellent screening tool magnetic resonance imaging (MRI) allows a comprehensive analysis of the heart in HCM. This includes a detailed analysis of the distribution and extent of myocardial hypertrophy, a thorough evaluation of systolic and diastolic cardiac function, the assessment of the presence and extent of dynamic outflow tract obstruction as well as the description of the systolic anterior motion (SAM) phenomenon of the mitral valve with secondary mitral insufficiency. When contrast material is administered, additional information about myocardial perfusion as well as the presence and extent of myocardial fibrosis can be obtained. This study compared systolic functional parameters as well as end systolic and end diastolic wall thickness of patients with and without diastolic dysfunction.

[A rest or arrest--asystolic episodes of 7 seconds in a 22-year-old athlete]. / 22-jähriger Sportler mit Sinusarrest und Pausen bis 7 Sekunden.

A 22-year-old athlete with nocturnal asymptomatic episodes of transient sinus arrest/sinoatrial block up to 7.3 s and recurrent inappropriate sinus tachycardias which had been incidentally found during Holter electrocardiography diagnostics is presented. In spite of extensive diagnostic work-up including invasive procedures like coronary angiography and electrophysiological study, no causal etiology was found. Based on the normal findings and the lack of symptoms, we decided not to implant a permanent pacemaker. After 14 months, the patient is still asymptomatic. Howerver, the 24-h Holter electrocardiography shows unchanged frequency of nocturnal transient sinus arrest episodes.

Acute respiratory distress syndrome with transiently impaired left ventricular function and Torsades de Pointes arrhythmia unmasking congenital long QT syndrome in a 25-yr-old woman.

We report a case of recurrent episodes of Torsades de Pointes arrhythmia in the setting of transiently impaired left ventricular ejection fraction, acute respiratory distress syndrome, transient hypokalaemia and QT-prolonging drugs, in a previously healthy 25-yr-old female patient. In the course of the clinical and genetic work-up this patient was newly diagnosed with a mutation in KCNH2 encoding the alpha-subunit of the human repolarizing potassium channel I(Kr). This case report illustrates the multivariate nature of long-QT syndrome, and emphasizes the usefulness of a pharmacological test for repolarization abnormalities.

Asymmetric reversal modes in ferromagnetic/antiferromagnetic multilayers.

Experimentally an asymmetry of the reversal modes has been found in certain exchange bias systems. From a numerical investigation of the domain state model evidence is gained that this effect depends on the angle between the easy axis of the antiferromagnet and the applied magnetic field. Depending on this angle the ferromagnet reverses either symmetrically, e.g., by a coherent rotation on both sides of the loop, or the reversal is asymmetric with a nonuniform reversal mode for the ascending branch, which may even yield a zero perpendicular magnetization.

An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome.

BACKGROUND: While the genome sequences for a variety of organisms are now available, the precise number of the genes encoded is still a matter of debate. For the human genome several stringent annotation approaches have resulted in the same number of potential genes, but a careful comparison revealed only limited overlap. This indicates that only the combination of different computational prediction methods and experimental evaluation of such in silico data will provide more complete genome annotations. In order to get a more complete gene content of the Drosophila melanogaster genome, we based our new D. melanogaster whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the Berkeley Drosophila Genome Project (BDGP) annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. RESULTS: Here we provide evidence for the transcription of approximately 2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicates a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. CONCLUSIONS: The successful design and application of this novel Drosophila microarray on the basis of our integrated in silico/wet biology approach confirms our expectation that in silico approaches alone will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or those that have not been strictly conserved between species.

Genetic identification of multiple susceptibility genes involved in the development of endometrial carcinoma in a rat model.

There are clear indications that inheritance plays an essential role in certain cases of human endometrial cancer, and there are at least 2 forms of early-onset heritable endometrial adenocarcinomas (EACs). Females of the BDII inbred rat strain are known to be genetically predisposed to endometrial carcinoma, and we have performed a genetic analysis of susceptibility to endometrial cancer in this strain. F(2) populations were generated by crossing BDII females with males from 2 different strains with a low incidence of EAC, and the occurrence of endometrial cancer was studied. Three chromosome regions associated to EAC susceptibility were identified, and the susceptibility genes in these regions were designated Ecs1, Ecs2 and Ecs3. Our results indicate that the genes affecting susceptibility to EAC are different in the 2 crosses, suggesting that the genes behind the susceptibility in BDII animals may interact with different genes in different genetic backgrounds.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization.

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Distribution of somatostatin receptor subtypes in rat lumbar spinal cord examined with gold-labelled somatostatin and anti-receptor antibodies.

Using gold-labelled somatostatin, somatostatin binding sites were predominantly found in laminae I-III, X and on motorneurones of the rat lumbar spinal cord. A comparison with immunohistochemical staining using antisera against somatostatin receptor sequences revealed that the marked binding in laminae I-III coincided with the presence of somatostatin receptor-like immunoreactivity for the receptor subtypes 1, 2 and 3. Binding sites on motorneurones were only paralleled by an immunoreaction for subtype 3. In lamina X, however, the lack of a positive immunoreaction indicates that in this part other subtypes may be present.

A new immunoenzymometric assay for bioactive N-terminal human parathyroid hormone fragments and its application in pharmacokinetic studies in dogs.

Advances in the treatment of clinical disorders of mineral in homeostatis and metabolic bone disease with intact parathyroid hormone 1-84 or one of the biologically active N-terminal fragments require a precise and sensitive measurement in serum. Therefore, a two-site immunoenzymometric assay for the quantitative determination of bioactive hPTH-1-37 (human parathyroid hormone) at picomolar concentrations was developed. Monoclonal antibodies (mAB) against hPTH-1-37 were raised by hybridoma cells in serum-free cell culture. Furthermore, sequence-specific polyclonal antibodies were obtained by immunisation of rabbits using multiple antigenic peptides (MAP) representing the conspicuous regions of the primary structure of hPTH-1-37. The polyclonal and monoclonal antibodies were characterised by epitope mapping. The combination of a monoclonal antibody (13C63/5) recognising hPTH fragment 16-24 with a polyclonal antibody (k2) showing a predominant binding sequence at hPTH-1-5 led to a sandwich assay specific for N-terminally intact and therefore biologically active hPTH. The validated assay ranging from 4 to 1000 pmol/l was applied to pharmacokinetic studies of hPTH-1-37. After s.c. administration of 30 mu g/kg in 5 beagles, the maximum serum concentrations of hPTH-1-37 ranging at 2139 +/- 857 pmol/l were observed 45 min after the injection. Clearance of the peptide calculated from the exponential disappearance curve was 32.0 +/- 9.1 ml/min/kg with a mean t1/2 of 37 +/- 10 min.

Effect of 17beta-estradiol-bisphosphonate conjugates, potential bone-seeking estrogen pro-drugs, on 17beta-estradiol serum kinetics and bone mass in rats.

In order to target 17beta-estradiol directly at bone we synthesized three 17beta-estradiol-bisphosphonate conjugates (E2-BPs) with different esterase-sensitive linkers between both molecular moieties. The systemic administration of these compounds should result primarily in local estrogenic effects on bone with no or negligible systemic hormonal effects. Only if a considerable margin exists between the doses required for inhibition of bone loss and those for systemic hormonal effects can such a pro-drug be considered acceptable for patients refusing systemic estrogen replacement therapy for several reasons. The conjugates were tested in vitro for their 17beta-estradiol release in rat serum and in vivo for their local and systemic effects in rats: in vitro, the conjugates expressed cleavage resistance, low cleavage (4.8%), or high cleavage (33.1%) within 48 hours of incubation. The conjugate with the low-cleavage doubled 17beta-estradiol serum half-life (3.78 hours) whereas the high-cleavage conjugate resulted in approximately four times higher serum half-life (8.36 hours) when compared with free 17beta-estradiol. In ovariectomized rats, bone loss was optimally prevented by 50 nmol/kg/day of 17beta-estradiol when administered S. C. over a period of 5 weeks, and protection against uterine atrophy was achieved at doses as low as 5 nmol/kg/day. The cleavage-resistant conjugate was ineffective in preserving bone and uterus in doses ranging from 5 to 150 nmol/kg/day. The other two E2-BPs revealed a dose-dependent inhibition of bone loss which was paralleled by the respective uterus weight with a dose range of 1.5-150 nmol/kg/day being fully effective in a range similar to 17beta-estradiol alone. The higher sensitivity of the uterus versus bone to protective estrogenic effects (1:10) was abolished by the conjugates. We conclude that E2-BPs containing esterase-sensitive linkers failed to act as bone-seeking pro-drugs expressing primarily local effects on bone without systemic effects.

Influence of the degree of renal dysfunction on the pharmacokinetic properties of the novel recombinant plasminogen activator reteplase in rats.

Reteplase (drug code: BM 06.022) is an unglycosylated recombinant plasminogen activator variant derived from human tissue-type plasminogen activator. The main metabolic organs of reteplase are the kidneys, liver, and blood, whereas human tissue-type plasminogen activator is predominantly cleared by the liver. Recent studies showed that reteplase plasma concentrations were significantly increased in severe acute renal failure. The purpose of the present study was to evaluate whether the degree of renal failure influences the pharmacokinetic properties of reteplase. Subacute renal failure in rats was induced by bilateral 1-hr clamping of the renal arteries and recovery for 3 or 6 days. Acute renal failure was induced by bilateral surgical nephrectomy. Renal function was assessed by inulin clearance. The plasma concentration of functionally active reteplase was measured by an indirect spectrophotometric assay. Reteplase was administered as a double-bolus intravenous injection of 140 + 140 kU/kg, 30 min apart. In comparison with sham surgery, 1-hr clamping plus recovery for 6 days had the least effect on inulin clearance, followed by clamping and recovery for 3 days and bilateral nephrectomy (20.2 +/- 1.8 vs. 13.0 +/- 1.3, 8.3 +/- 0.8, and 3.1 +/- 0.2 ml center dot min-1 center dot kg-1, p < 0.01). Total plasma clearance of reteplase was significantly reduced, compared with sham surgery after 1-hr clamping plus 3-day recovery and bilateral nephrectomy (3.65 +/- 0.26 vs. 2.6 +/- 0.23 and 2.18 +/- 0.14 ml center dot min-1 center dot kg-1, p < 0.05 and p < 0.01, respectively), but not after 1-hr clamping plus 6-day recovery (3.33 +/- 0.34 ml center dot min-1 center dot kg-1, NS vs. sham surgery). There was a significant (p < 0.0001) linear correlation (r = 0.713) between the decrease of inulin clearance and the decrease of reteplase clearance. These data indicate that slight impairment of renal function does not significantly influence pharmacokinetic properties of reteplase, whereas severe renal dysfunction does.

Parathyroid hormone increases the concentration of insulin-like growth factor-I and transforming growth factor beta 1 in rat bone.

Intermittent treatment with parathyroid hormone (PTH) increases bone mass in experimental animals and humans. In vitro studies have suggested that the anabolic effect of PTH may be mediated by local growth factors. However, the relevance of these findings to in vivo situations remains unclear. In this study, we examined a time course of daily s.c. injections of hPTH (1-34) on the skeletal concentration of insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor beta (TGF-beta) in the proximal tail vertebrae of male rats. PTH caused a time and dose-dependent increase in the bone mineral density of the lumbar spine. This anabolic effect on bone mass was accompanied by progressive increases in bone matrix-associated IGF-I and TGF-beta 1. Increases in IGF-I and TGF-beta 1 became apparent after four and eight weeks of PTH treatment respectively and persisted through week 12. PTH had no effect on circulating IGF-I, suggesting that the increase of bone matrix IGF-I was due to the local effect of PTH on bone tissue directly rather than to an increase of circulating IGF-I. These data are consistent with the hypothesis that IGF-I and TGF-beta 1 may play a role as local mediators of the anabolic effects of PTH on bone metabolism.

Naftopidil, a new adrenoceptor blocking agent with Ca(2+)-antagonistic properties: interaction with adrenoceptors.

The interaction of naftopidil with adrenoceptors was studied in comparison to standard drugs. Naftopidil binds specifically to alpha 1-adrenoceptors. The Ki values are 58.3 nM for naftopidil, 0.43 nM for prazosin, and 197 nM for urapidil. The affinities of naftopidil to alpha 2- and beta-adrenoceptor sites are very low (> 6,000 and > 2,500 nM). Naftopidil relaxes aortic strips precontracted with norepinephrine concentration-dependently, and it shifts the concentration-response curve of norepinephrine in a parallel manner to the right. The pA2 values are 7.10 for naftopidil, 8.85 for prazosin, and 6.25 for urapidil. In pithed rats, naftopidil shifted the dose-response curve of methoxamine at equipotent hypotensive doses to the same extent to the right as does prazosin, but both drugs barely affected (in contrast to phentolamine) the response to norepinephrine. In concentrations that are about 10 times higher than those required for alpha 1-adrenoceptor blockade, naftopidil relaxes (in contrast to prazosin) aortic strips depolarized with K+, and it shifts Ca2+ concentration-response curves to the right (pA2 value of 5.90), thus suggesting Ca(2+)-channel-blocking activity. Both alpha-adrenoceptor and Ca(2+)-blocking activities are exerted to nearly the same extent by both stereoisomers. Naftipidil does not affect the response to isoprenaline-induced effects, indicating that the compound does not possess beta-blocking properties.

Effects of carvedilol on adrenergic receptor pharmacology in human ventricular myocardium and lymphocytes.

Carvedilol, a new beta-blocker with vasodilating properties due to alpha 1-blockade, was investigated in preparations of human ventricular myocardium. Carvedilol demonstrated a high affinity and is a slightly beta 1-selective competitive beta-blocking agent, with a KD for beta 1-receptors of approximately 4-5 nM and a mild selectivity for beta 1 vs. beta 2 receptors of 6- to 39-fold, depending on the method employed to assess subtype potency. In addition, carvedilol was also a potent alpha 1-blocking agent, with a beta 1:alpha 1 blocking relative potency of 1.7-fold. In human lymphocytes containing beta 2-receptors and in human myocardial membranes containing both beta 1- and beta 2-receptors carvedilol exhibited the unique property of guanine nucleotide modulatable binding. Despite this, no intrinsic sympathomimetic activity of carvedilol was detected in preparations of isolated human heart or in myocardial membranes. Vasodilation related to alpha 1-blockade and the lack of intrinsic activity should translate into improved tolerability and good efficacy in the treatment of heart failure.

Effects of carvedilol on ventricular arrhythmias.

Carvedilol is a nonselective beta-blocker with alpha-mediated vasodilating properties that has been shown to be effective in systemic hypertension, stable angina, and congestive heart failure (CHF). In this study, we studied the effects of carvedilol on premature ventricular contractions (PVCs) in 65 patients undergoing treatment with carvedilol (12.5-50 mg b.i.d.) for 4-8 weeks. Twelve patients had hypertension, 41 had stable angina, and 12 had CHF. Holter monitoring for 24 h was performed before and after active carvedilol therapy. Median PVCs per 24 h decreased from 25.5 to 6.0 (p less than 0.001, n = 52). Reduction in PVC activity occurred in 77% of patients, and 23% of patients with multifocal PVCs changed their morphology to unifocal. Nonsustained ventricular tachycardia was present in four patients before treatment; this was abolished in all patients. R-on-T PVC was present in six patients; it decreased in five and increased in one. New ventricular tachycardia (less than 8 beats) occurred in two patients, but QT prolongation was not noted in these patients. An improvement in Lown's classification occurred in 50% of patients. However, in the CHF group, the improvement in Lown's criteria was observed in 73% of patients. Carvedilol does not appear to possess proarrhythmic effects, and chronic therapy reduces PVC activity in a wide range of patients. This property of carvedilol is complementary to its hypotensive and anti-ischemic effects. In the CHF group, the beneficial effects of carvedilol on left ventricular function and hemodynamics may combine with the improvement in PVC activity to produce a significant improvement in mortality.

Vasodilatory action of carvedilol.

Carvedilol is a dual-acting drug designed to produce two complementary effects: beta-blockade and vasodilation. These effects are induced in the same dose range, a prerequisite for utilizing both properties in an appropriate manner. The vasodilation is mediated predominantly by specific alpha 1-adrenoceptor blockade. At markedly higher concentrations, additional vasodilating actions besides alpha 1-blockade can be observed. These effects resemble those of Ca(2+)-antagonistic properties. However, they do not contribute to the acute blood pressure-lowering activity of carvedilol but may be responsible for the increased blood flow to specific organs. At beta-blocking doses, carvedilol reduces the regional and systemic vascular resistance in various experimental models, healthy volunteers, and in patients with cardiovascular diseases such as hypertension, coronary artery disease, and heart failure. The profile of carvedilol thus insures beneficial treatment of hemodynamic disorders characterized by increased sympathetic tone and increased vascular resistance.

Receptor pharmacology of carvedilol in the human heart.

The beta-blocker and vasodilator carvedilol was examined in preparations of human ventricular myocardium. Carvedilol is a high-affinity, slightly beta 1-selective competitive beta-blocking agent, with a KD for beta 1-receptors of approximately 4-5 nM and a selectivity of sixfold to 39-fold for beta 1-receptors rather than beta 2-receptors, depending on the method used to assess subtype potency. Carvedilol also is a potent alpha 1-blocking agent, with a beta 1: alpha 1-blocking relative potency of 1.7-fold. In human lymphocytes containing beta 2-receptors and human myocardial membranes containing both beta 1- and beta 2-receptors, carvedilol exhibited the unique property of guanine nucleotide-modulatable binding. This is a property shared with bucindolol, another beta-blocker and vasodilator that is structurally similar to carvedilol. Despite the presence of guanine nucleotide-modulatable binding, no intrinsic activity of carvedilol was detected in preparations of isolated human heart or in myocardial membranes.

Haemodynamic profile of an inhibitor of phosphodiesterase III, adibendan (BM 14.478): comparison with nitroprusside and dobutamine in conscious dogs.

1. This study was performed to investigate whether cardiac positive inotropic as well as peripheral vasodilator properties of adibendan contribute to its overall haemodynamic profile in conscious dogs. 2. Haemodynamic measurements were carried out in conscious chronically instrumented dogs after administration of adibendan, sodium nitroprusside or dobutamine. 3. The cardiovascular changes induced by adibendan (0.01 and 0.03 mg kg-1) resembled those of dobutamine (1.0-4.0 micrograms kg-1 min-1): left ventricular dP/dt60 (LV dP/dt60), stroke volume (SV) and cardiac output (CO) increased to a similar extent, but mean arterial pressure (MAP) and heart rate (HR) remained unchanged. 4. In contrast to dobutamine, higher doses of adibendan (0.1-1.0 mg kg-1) decreased MAP and LVEDP. These effects were of a similar magnitude to those observed following nitroprusside administration (0.5-12.5 micrograms kg-1 min-1). In contrast to nitroprusside, adibendan still showed additional effects on LV dP/dt60 and CO. 5. From these results, it is concluded that both the peripheral vasodilator and the cardiac positive inotropic action of adibendan contribute to its overall haemodynamic profile.

Nonsteroidal cardiotonics. 3. New 4,5-dihydro-6-(1H-indol-5-yl)pyridazin-3(2H)-ones and related compounds with positive inotropic activities.

A series of substituted indolyldihydropyridazinones and related compounds 1-18 were synthesized and evaluated for positive inotropic activity. In rats, most of these indole derivatives produced a dose-related increase in myocardial contractility with little effect on heart rate and blood pressure. Compound 13, 4,5-dihydro-5-methyl-6-(2-pyridin-4-yl-1H-indol-5-yl)pyrazin-3(2H) -one (BM 50.0430), was further investigated in cats. The increase in contractility in this animal model was not mediated via stimulation of beta-adrenergic receptors. After oral administration of 1 mg/kg to conscious dogs, compound 13 and pimobendan were still active after 6.5 h. However, the cardiotonic effect of 13 was at least 2-fold that of pimobendan after this period of time. The structural requirements necessary for optimal cardiotonic activity within this novel class of indole derivatives are a heterocyclic aromatic ring in position 2, a hydrogen or a methyl group in position 3, and a dihydropyridazinone ring system in position 5 of the indole.