Biomedical subjects

Juliane Stieber

Publications and source records attributed to Juliane Stieber.

7 recordsLinked to original sources

PubMed2005

Bradycardic and proarrhythmic properties of sinus node inhibitors.

Sinus node inhibitors reduce the heart rate presumably by blocking the pacemaker current If in the cardiac conduction system. This pacemaker current is carried by four hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels. We tested the potential subtype-specificity of the sinus node inhibitors cilobradine, ivabradine, and zatebradine using cloned HCN channels. All three substances blocked the slow inward current through human HCN1, HCN2, HCN3, and HCN4 channels. There was no subtype-specificity for the steady-state block, with mean IC50 values of 0.99, 2.25, and 1.96 microM for cilobradine, ivabradine, and zatebradine, respectively. Native If, recorded from mouse sinoatrial node cells, was slightly more efficiently blocked by cilobradine (IC50 value of 0.62 microM) than were the HCN currents. The block of I(f) in sinoatrial node cells resulted in slower and dysrhythmic spontaneous action potentials. The in vivo action of these blockers was analyzed using telemetric ECG recordings in mice. Each compound reduced the heart rate dose-dependently from 600 to 200 bpm with ED50 values of 1.2, 4.7, and 1.8 mg/kg for cilobradine, ivabradine, and zatebradine, respectively. beta-Adrenergic stimulation or forced physical activity only partly reversed this bradycardia. In addition to bradycardia, all three drugs induced increasing arrhythmia at concentrations greater than 5 mg/kg for cilobradine, greater than 10 mg/kg for zatebradine, or greater than 15 mg/kg for ivabradine. This dysrhythmic heart rate is characterized by periodic fluctuations of the duration between the T and P wave, resembling a form of sick sinus syndrome in humans. Hence, all available sinus node inhibitors possess an as-yet-unrecognized proarrhythmic potential.

Animals↗

PubMed2005

Functional expression of the human HCN3 channel.

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlie the inward pacemaker current, termed I(f)/I(h), in a variety of tissues. Many details are known for the HCN subtypes 1, 2, and 4. We now successfully cloned the cDNA for HCN3 from human brain and compared the electrophysiological properties of hHCN3 to the other three HCN subtypes. Overexpression of human HCN3 channels in HEK293 cells resulted in a functional channel protein. Similar to hHCN2 channels, hHCN3 channels are activated with a rather slow time constant of 1244 +/- 526 ms at -100 mV, which is a greater time constant than that of HCN1 but a smaller one than that of HCN4 channels. The membrane potential for half-maximal activation V((1/2)) was -77 +/- 5.4 mV, and the reversal potential E(rev) was -20.5 +/- 4 mV, resulting in a permeability ratio P(Na)/P(K) of 0.3. Like all other HCNs, hHCN3 was inhibited rapidly and reversibly by extracellular cesium and slowly and irreversibly by extracellular applied ZD7288. Surprisingly, the human HCN3 channel was not modulated by intracellular cAMP, a hallmark of the other known HCN channels. Sequence comparison revealed >80% homology of the transmembrane segments, the pore region, and the cyclic nucleotide binding domain of hHCN3 with the other HCN channels. The missing response to cAMP distinguishes human HCN3 from both the well cAMP responding HCN subtypes 2 and 4 and the weak responding subtype 1.

Blotting, Northern↗

PubMed2004

IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase.

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.

Animals↗

PubMed2004

Pacemaker channels and sinus node arrhythmia.

Cardiac pacemaker activity is regulated by at least five different classes of ion channels and by the opposing influence of sympathetic and parasympathetic stimulation. Inactivation of several genes, including a subunit coding for the potassium channel activated by the muscarinic receptor, I(KACh); the calcium channel, I(Ca,); and the hyperpolarization-activated channel, I(f), results in sinus node arrhythmia. Inactivation of the gene for the hyperpolarization-activated, cyclic nucleotide-gated channel isoform HCN2 or HCN4 and the use of pacemaker channel blockers show that (a) HCN2 prevents the diastolic membrane potential from becoming too negative, (b) HCN4 is the major channel mediating sympathetic stimulation of the pacemaker activity, and (3) complete blockage of the I(f) current is compatible with slow sinus node rhythm.

Action Potentials↗

PubMed2003

The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart.

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with "primitive" pacemaker action potentials could be found. However, cardiac cells with "mature" pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.

Action Potentials↗

PubMed2003

Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4.

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (tau act), which is fast for HCN2 (144 ms at -140 mV) and slow for HCN4 (461 ms at -140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased tau act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted tau act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in tau act. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.

Amino Acid Sequence↗

PubMed2003

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.

Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1-4) to these diverse functions are not known. Here we show that HCN2-deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte- specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Animals↗