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Ursula Ravens

Publications and source records attributed to Ursula Ravens.

At least 37 records · Page 2Linked to original sources
PubMed2005

Risperidone-induced action potential prolongation is attenuated by increased repolarization reserve due to concomitant block of I(Ca,L).

The neuroleptic risperidone is an effective blocker of the rapidly activating component of the delayed rectifier current (I(Kr)) and hence is expected to prolong cardiac action potential duration (APD). However, unlike with other typical I(Kr) blockers we failed to demonstrate a marked prolongation of late repolarization with risperidone. It is hypothesized that the APD-prolonging effect of risperidone is masked by the high repolarization reserve due to the prominent delayed rectifier currents I(Kr) and I(Ks) in guinea pig papillary muscle. Action potentials and force of contraction were recorded in isolated guinea pig papillary muscles. L-type calcium current I(Ca,L) and I(Kr) were measured using the standard patch clamp technique in single ventricular cardiomyocytes. Reduction of the repolarization reserve by the blocking of I(Ks) with chromanol 239B augmented the effect of the selective I(Kr) blocker E-4031, but not of risperidone, although both drugs completely blocked I(Kr). In contrast to E-4031 risperidone markedly reduced the force of contraction due to the partial blocking of I(Ca,L) in the same concentration range as required for block of I(Kr). Reduction of the repolarization reserve by the blocking of I(Ks) cannot exacerbate the APD-prolonging effect of risperidone. However, even incomplete concomitant blocking of I(Ca,L) attenuates the APD-prolonging effect of the complete blocking of I(Kr). This behaviour may explain the small APD-prolonging effect of risperidone despite the drug's robust blocking of I(Kr).

Action Potentials↗
PubMed2005

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

UNLABELLED: We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. INTRODUCTION: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. MATERIALS AND METHODS: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. RESULTS: mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. CONCLUSION: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Adult↗
PubMed2005

Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.

Dipeptidyl-aminopeptidase-like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A-type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (I(to)) are not mimicked by coexpression of the alpha-subunit Kv4.3 with its known beta-subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional beta-subunit in the human heart. With quantitative real-time RT-PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX-specific antibody. Co-expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I(to)-like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half-maximum steady-state inactivation was more negative and recovery from inactivation was delayed. Co-expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I(to) channel complex in human heart.

Amino Acid Sequence↗
PubMed2005

Silencing the cardiac potassium channel Kv4.3 by RNA interference in a CHO expression system.

RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by short RNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was to test the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order to investigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessory beta-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at the mRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3 mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outward current (I(to)) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional gene silencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adapted for studying their role also in native cells.

Animals↗
PubMed2005

Defective cardiac ryanodine receptor regulation during atrial fibrillation.

BACKGROUND: Ca2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. METHODS AND RESULTS: Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca2+ leak. CONCLUSIONS: SR Ca2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Animals↗
PubMed2005

Tissue distribution of a human Ca v 1.2 alpha1 subunit splice variant with a 75 bp insertion.

Mesenchymal stem cells from human bone marrow (MSC) express mRNA encoding the L-type Ca2+ channel Ca v 1.2 alpha1 subunit (alpha(1)1.2). We now describe a splice variant including an alternative exon of 75 bp in the region between exons 9 and 10, which we identified in MSC by semi-quantitative RT-PCR. With primers specific for variants including (+9*) or excluding the 75 bp insertion (-9*), we found comparable mRNA expression patterns in MSC and in primary cultures of related connective tissue cells (chondrocytes, osteoblasts and fibroblasts). Since culture conditions might have altered variant expression, we investigated mRNA levels in various native human tissue samples (cartilage, bone, fat, liver, kidney, aorta, bladder, cardiac ventricle and atrium, CNS). We found highest levels of the +9* variant in aorta, containing smooth muscle and connective tissue cells, but the variant was expressed in all tissues. We therefore hypothesized that broad expression of +9* might be linked to the presence of vasculature and/or connective tissue structures, rather than to tissue-specific parenchymal cells (e.g. cardiomyocytes). To test this hypothesis we separated human atrium into a cardiomyocyte-enriched fraction and a cardiomyocyte-depleted fraction. RT-PCR demonstrated significantly larger levels of the +9* variant in the non-cardiomyocyte fraction. The result was even more clear in single cell RT-PCR experiments, where the +9* variant was undetectable in cardiomyocytes but present in non-cardiomyocytes. We conclude that the +9* variant is present in all human tissues investigated so far, and suggest that expression in human atrium is associated with vascular smooth muscle and/or connective tissue cells.

Adult↗
PubMed2005

Comparing the global mRNA expression profile of human atrial and ventricular myocardium with high-density oligonucleotide arrays.

OBJECTIVES: The knowledge of chamber-specific gene expression in human atrial and ventricular myocardium is essential for the understanding of myocardial function and the basis for the identification of putative therapeutic targets in the treatment of cardiac arrhythmia and heart failure. In this study the gene expression pattern of human left atrial and ventricular myocardium was analyzed. METHODS: Global mRNA expression patterns with high-density oligonucleotide arrays between left atrial and left ventricular myocardium of 6 patients with heart failure undergoing heart transplantation were compared. Clustering of microarray data confirmed chamber-specific gene expression profiles. Genes similarly expressed in all patients were further analyzed, and data were confirmed by means of real-time polymerase chain reaction and Western blot analysis. RESULTS: Of 22,215 genes examined, 7115 transcripts were found to be expressed in all 12 human myocardial samples. One hundred twenty-five genes were differentially expressed between left atrial and left ventricular specimens in all patients examined. Novel genes preferentially expressed in human atria were identified. Interestingly, several potassium channels of subfamily K are more highly expressed in atria than in ventricles. Members of the potassium inwardly rectifying channel of subfamily J were found to be more highly expressed in human ventricular myocardium. Finally, chronic atrial fibrillation was associated with reduced atrial expression of the potassium channel TWIK-1, suggesting potential contribution of the corresponding current to electrical remodeling. CONCLUSIONS: Human atria and ventricles show specific gene expression profiles. Our data provide the basis of a comprehensive understanding of chamber-specific gene expression in diseased human hearts and will support the identification of therapeutic targets in the treatment of arrhythmia and heart failure.

Gene Expression Profiling↗
PubMed2005

Pharmacodynamics of propiverine and three of its main metabolites on detrusor contraction.

1. Besides its antimuscarinic effects, propiverine may possess an additional mode of action. We compared the effects of propiverine, three of its metabolites (M-5, M-6, M-14) and atropine in human, pig and mouse urinary bladder preparations in order to elucidate the nature of a possible additional mode of action. 2. Like the parent compound, M-5, M-6 and M-14 reduced to variable degrees the contractions elicited by electric field stimulation (EFS) of isolated, urothelium-denuded detrusor strips. In mouse the atropine-resistant and therefore the nonadrenergic, noncholinergic component of contractile response to EFS was reduced by M-5, M-14 and propiverine, but was hardly affected by M-6. 3. Atropine, propiverine and M-6 significantly shifted the cumulative concentration-response curves for carbachol (CCh) to higher concentrations. Atropine and M-6 did not affect the maximum tension induced by CCh. Propiverine, M-5 and M-14 reduced the maximum CCh effect, suggesting at least one additional mode of action. This pattern of response was observed in all the three species, albeit with some differences in sensitivity to the various agents. 4. In freshly isolated human detrusor smooth muscle cells, propiverine and M-14 inhibited the nifedipine-sensitive L-type calcium current (I(Ca)) in a concentration-dependent manner. In contrast, the effects of M-5 and M-6 on I(Ca) were insignificant in the concentration range examined. 5. The investigated responses to propiverine and its metabolites suggest that impairment of maximum CCh-induced contractions is due to strong effect on I(Ca) and that this may be associated with the presence of the aliphatic side chain.

Aged↗
PubMed2005

Cholinergic and purinergic responses in isolated human detrusor in relation to age.

PURPOSE: We investigated whether the contractility of isolated human detrusor muscle, responsiveness to commonly used spasmolytic drugs, and expression of selected muscarinic and purinergic (P2X) receptor subtypes (M2, M3, P2X1 and P2X3) change with age. MATERIALS AND METHODS: Tissues were taken from 63 patients 37 to 84 years old undergoing radical cystectomy. Specimens from 49 patients were used for contractility studies and those from 50 were used for mRNA analysis. RESULTS: Propiverine, oxybutynin, tolterodine and atropine decreased contractions evoked by electric field stimulation to different degrees. However, neither the efficacy nor potency of the drugs showed age related changes. Since human detrusor muscle shows atropine resistant noncholinergic responses, we also studied the putative age dependence of concentration-response curves to the muscarinic agonist carbachol, and the purinergic agonists adenosine triphosphate (ATP) and alpha-beta-methylene-ATP. Sensitivity to alpha-beta-methylene-ATP increased with age, while the efficacy and potency of spasmolytic drugs did not depend on age. In addition, mRNA detected for M2, M3, P2X1 and P2X3 receptors did not change with age. CONCLUSIONS: Our results do not provide evidence for age related contractile deterioration in human detrusor muscle strips, nor do they suggest that responses to anticholinergic spasmolytic drugs change substantially with age.

Adult↗
PubMed2005

Effect of local anaesthesia on neuronal c-fos expression in the spinal dorsal horn and hypothalamic paraventricular nucleus after surgery in rats.

The surgical stress response is the neurophysiologic reflex response to surgery, which involves activation of the hypothalamic-pituitary-adrenal axis and is regulated by the hypothalamic paraventricular nucleus. The effect of pre-operative use of local anaesthetics on activation of neurones in the paraventricular nucleus during surgery was studied by quantification of the neuronal expression of the c-fos-gene after a standardized plantar incision in rats. Furthermore, c-fos expression in the spinal dorsal horn was used as a measure of spinal nociception. Six halothane-anaesthetized animals underwent surgery following infiltration with lidocaine and bupivacaine, six animals were operated without local anaesthetics, and six control animals were subjected to the anaesthetic procedures. After two hours, the animals were perfused with 4% formaldehyde and the spinal cords and brains were collected and processed by immunohistochemistry for stereological quantification of the number of neurones with Fos-like immunoreactivity. Furthermore, brain and spinal cord were sampled from nine control animals right after induction of halothane anaesthesia. Surgery without local anaesthetics caused a significant increased number of neurones with Fos-like immunoreactivity in the spinal cord (4258+/-1710; mean+/-S.D.; P<0.01) compared to the anaesthesia control group (1204+/-436). Local anaesthetics reduced this number to 2029+/-919 (P<0.05), which was not significantly different from the anaesthesia control group. After surgery, the number of neurones with Fos-like immunoreactivity in paraventricular nucleus increased from 2948+/-1365 in the anaesthetized control group to 5550+/-3875 and 5191+/-1558 in the surgery and local anaesthetics plus surgery group, respectively, although significance was only reached for the group receiving local anaesthetics (P<0.05). In conclusion, preoperative local anaesthetic infiltration did not reduce the surgery-induced c-fos expression in paraventricular nucleus after paw surgery in rats, although spinal nociception was reduced.

Anesthetics, Local↗
PubMed2004

Beta adrenergic receptor-mediated atrial specific up-regulation of RGS5.

Previous investigations had suggested that signaling from the overexpressed beta(2) adrenergic in the heart of transgenic TG4 mice was dampened in the atria. Using an RT-PCR based strategy, we have identified Regulator of G-protein Signaling 5 (RGS5) as being up-regulated in the atria of TG4 mice. Northern blot analysis demonstrated that RGS5 levels were 3 fold higher in the atria of TG4 mice. Western blot analysis of a panel of rat tissues demonstrated that basal expression of RGS5 protein was confined to the heart and skeletal muscle. Furthermore, RGS5 protein was detected in skeletal muscle C2C12 and cardiomyocyte HL-1 cultured cell lines. As observed for RGS5 mRNA levels in TG4 mice, RGS5 protein levels were increased in the atria of rats that were administered the beta adrenergic agonist isoproterenol during a 14 day period. Taken together, these results indicate that RGS5 is a housekeeping RGS in the heart and in skeletal muscle while its beta adrenergic-mediated induction in the atrium suggests that it also has a highly specialized function.

Adrenergic beta-Agonists↗
PubMed2004

An aqueous extract of the marine sponge Ectyoplasia ferox stimulates L-type Ca2+-current by direct interaction with the Cav1.2 subunit.

Marine organisms have attracted much attention as a source of pharmacological tools or potential drugs. We have produced and screened a library of sponge extracts in search of biologically active compounds that may contain useful pharmaceutical lead structures. Sponges were collected from various locations and their aqueous extracts were freeze dried. Murine right and left atria were used to screen 75 extracts for putative cardiac effects. Among seven extracts with a positive inotropic and chronotropic effect the extract C47 from Ectyoplasia ferox proved to be the most active and was chosen for further analysis. C47 also produced a beta-adrenoceptor-independent, propranolol-resistant positive inotropic effect in human atrial trabeculae. To elucidate one possible mode of action the effects of C47 on L-type Ca(2+) current (I(Ca,L)) were measured with a standard patch-clamp technique. In isolated human atrial myocytes exposure to C47 increased peak amplitude of I(Ca,L) in a concentration-dependent manner. The threshold concentration was 15 microg/ml. In addition, voltage dependency of activation and steady-state inactivation were shifted to more negative potentials. C47 slowed the initial phase of time-dependent current inactivation and the recovery from inactivation. In cell-attached patches of HEK 293 cells expressing human Ca(v)1.2 addition of C47 to the bath solution did not affect gating properties, whereas inclusion of the extract into the pipette solution strongly increased single-channel activity, suggesting a direct effect on the pore-forming channel subunit. Despite its robust effect on I(Ca,L) C47 enhanced cardiac force of contraction by only a fraction of the maximum increase caused by high extracellular concentrations of Ca(2+) and failed to increase vascular tone. These findings suggest that the effect of C47 is restricted to the Ca(2+) channel.

Action Potentials↗
PubMed2004

Role of IKur in controlling action potential shape and contractility in the human atrium: influence of chronic atrial fibrillation.

BACKGROUND: The ultrarapid outward current I(Kur) is a major repolarizing current in human atrium and a potential target for treating atrial arrhythmias. The effects of selective block of I(Kur) by low concentrations of 4-aminopyridine or the biphenyl derivative AVE 0118 were investigated on right atrial action potentials (APs) in trabeculae from patients in sinus rhythm (SR) or chronic atrial fibrillation (AF). METHODS AND RESULTS: AP duration at 90% repolarization (APD90) was shorter in AF than in SR (300+/-16 ms, n=6, versus 414+/-10 ms, n=15), whereas APD20 was longer (35+/-9 ms in AF versus 5+/-2 ms in SR, P<0.05). 4-Aminopyridine (5 micromol/L) elevated the plateau to more positive potentials from -21+/-3 to -6+/-3 mV in SR and 0+/-3 to +12+/-3 mV in AF. 4-Aminopyridine reversibly shortened APD90 from 414+/-10 to 350+/-10 ms in SR but prolonged APD90 from 300+/-16 to 320+/-13 ms in AF. Similar results were obtained with AVE 0118 (6 micromol/L). Computer simulations of I(Kur) block in human atrial APs predicted secondary increases in I(Ca,L) and in the outward rectifiers I(Kr) and I(Ks), with smaller changes in AF than SR. The indirect increase in I(Ca,L) was supported by a positive inotropic effect of 4-aminopyridine without direct effects on I(Ca,L) in atrial but not ventricular preparations. In accordance with the model predictions, block of I(Kr) with E-4031 converted APD shortening effects of I(Kur) block in SR into AP prolongation. CONCLUSIONS: Whether inhibition of I(Kur) prolongs or shortens APD depends on the disease status of the atria and is determined by the level of electrical remodeling.

4-Aminopyridine↗
PubMed2004

Pharmacological modulation of ion channels and transporters.

Ion channels and transporter proteins are prerequisites for formation and conduction of cardiac electrical impulses. Acting in concert, these proteins maintain cellular Na(+) and Ca(2+) homeostasis. Since intracellular Ca(2+) concentration determines contractile activation, we expect the majority of agents that modulate activity of ion channels and transporters not only to influence cellular action potentials but also contractile force. Drugs which block ion channels usually possess antiarrhythmic properties, those inhibiting the Na(+) pump have predominantly inotropic effects and those affecting Na(+),Ca(2+)- or Na(+),H(+)-exchanger protect against ischaemic cell damage. However, irrespective of their primary indication, all compounds targeted against ion channels and transporter proteins possess potential proarrhythmic activity.

Animals↗
PubMed2004

Recording atrial monophasic action potentials using standard pacemaker leads: an alternative way to study electrophysiology properties of the human atrium in vivo?

AF leads to electrophysiological changes, but it is not known if similar alterations also appear before the onset of the first episode of AF because invasive electrophysiological studies are not justified in otherwise symptom-free patients. To address this question requires a safe method of obtaining atrial electrophysiological parameters at no extra risk or discomfort for the patient. The aim of this study was to test if recording of monophasic action potentials (MAPs) is feasible during pacemaker implantation. The study included 22 patients undergoing pacemaker implantation for symptomatic bradycardia without any history of AF. Using a custommade amplifier and a minor modification of the routine procedure for intraoperatively measured P waves, atrial electrograms could be recorded using a standard active pacemaker lead. MAP-like electrograms were obtained in 15 patients. MAP amplitude was 2.6 +/- 0.3 mV, mean action potential duration was 316 +/- 12 ms at a spontaneous heart rate of 67.2 +/- 3.2 beats/min. MAP duration was decreased when atria were stimulated at shorter cycle lengths (249 +/- 12 ms at 150 beats/min, P <0.05 vs sinus rhythm). In about two thirds of patients undergoing pacemaker implantation, recording of MAP-like electrograms was feasible with only minor modification of the atrial electrogram recording technique. The method should allow screening patients for electrophysiological alterations even before the onset of AF.

Aged↗