The complex molecular mechanisms underlying spontaneous cardiac pacemaking are not fully understood. Recent findings point to a co-ordinated interplay between intracellular Ca(2+) cycling and plasma ...membrane-localized cation transport determining the origin and periodicity of pacemaker potentials. The sodium-calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Here, we investigated the contribution of NCX1 to cardiac pacemaking.
We used an inducible and sinoatrial node-specific Cre transgene to create micelacking NCX1 selectively in cells of the cardiac pacemaking and conduction system (cpNCX1KO). RT-PCR and immunolabeling experiments confirmed the precise tissue-specific and temporally controlled deletion. Ablation of NCX1 resulted in a progressive slowing of heart rate accompanied by severe arrhythmias. Isolated sinoatrial tissue strips displayed a significantly decreased and irregular contraction rate underpinning a disturbed intrinsic pacemaker activity. Mutant animals displayed a gradual increase in the heart-to-body weight ratio and developed ventricular dilatation; however, their ventricular contractile performance was not significantly affected. Pacemaker cells from cpNCX1KO showed no NCX1 activity in response to caffeine-induced Ca(2+) release, determined by Ca(2+) imaging. Regular spontaneous Ca(2+) discharges were frequently seen in control, but only sporadically in knockout (KO) cells. The majority of NCX1KO cells displayed an irregular and a significantly reduced frequency of spontaneous Ca(2+) signals. Furthermore, Ca(2+) transients measured during electrical field stimulation were of smaller magnitude and decelerated kinetics in KO cells.
Our results establish NCX1 as a critical target for the proper function of cardiac pacemaking.
Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed Ifor 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, Ifin 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.
Several studies implicated cyclic adenosine monophosphate (cAMP) as an important second messenger for regulating nociceptor sensitization, but downstream targets of this signaling pathway which ...contribute to neuronal plasticity are not well understood. We used a Cre/loxP-based strategy to disable the function of either HCN2 or PKA selectively in a subset of peripheral nociceptive neurons and analyzed the nociceptive responses in both transgenic lines. A near-complete lack of sensitization was observed in both mutant strains when peripheral inflammation was induced by an intradermal injection of 8br-cAMP. The lack of HCN2 as well as the inhibition of PKA eliminated the cAMP-mediated increase of calcium transients in dorsal root ganglion neurons. Facilitation of Ih via cAMP, a hallmark of the Ih current, was abolished in neurons without PKA activity. Collectively, these results show a significant contribution of both genes to inflammatory pain and suggest that PKA-dependent activation of HCN2 underlies cAMP-triggered neuronal sensitization.
RATIONALE:The hyperpolarization-activated current Ih that is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) plays a key role in the control of pacemaker activity in ...sinoatrial node cells of the heart. By contrast, it is unclear whether Ih is also relevant for normal function of cardiac ventricles.
OBJECTIVE:To study the role of the HCN3-mediated component of ventricular Ih in normal ventricular function.
METHODS AND RESULTS:To test the hypothesis that HCN3 regulates the ventricular action potential waveform, we have generated and analyzed a HCN3-deficient mouse line. At basal heart rate, mice deficient for HCN3 displayed a profound increase in the T-wave amplitude in telemetric electrocardiographic measurements. Action potential recordings on isolated ventricular myocytes indicate that this effect was caused by an acceleration of the late repolarization phase in epicardial myocytes. Furthermore, the resting membrane potential was shifted to more hyperpolarized potentials in HCN3-deficient mice. Cardiomyocytes of HCN3-deficient mice displayed approximately 30% reduction of total Ih. At physiological ionic conditions, the HCN3-mediated current had a reversal potential of approximately −35 mV and displayed ultraslow deactivation kinetics.
CONCLUSIONS:We propose that HCN3 together with other members of the HCN channel family confer a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization. In conclusion, our data indicate that HCN3 plays an important role in shaping the cardiac action potential waveform.
Therapeutic approaches for "sick sinus syndrome" rely on electrical pacemakers, which lack hormone responsiveness and bear hazards such as infection and battery failure. These issues may be overcome ...via "biological pacemakers" derived from pluripotent stem cells (PSCs). Here, we show that forward programming of PSCs with the nodal cell inducer TBX3 plus an additional Myh6-promoter-based antibiotic selection leads to cardiomyocyte aggregates consisting of >80% physiologically and pharmacologically functional pacemaker cells. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300-400 bpm), coming close to those found in mouse hearts, and were able to robustly pace myocardium ex vivo. Our study introduces iSABs as highly pure, functional nodal tissue that is derived from PSCs and may be important for future cell therapies and drug testing in vitro.
Functional Expression of the Human HCN3 Channel Stieber, Juliane; Stöckl, Georg; Herrmann, Stefan ...
Journal of biological chemistry/The Journal of biological chemistry,
10/2005, Letnik:
280, Številka:
41
Journal Article
Recenzirano
Odprti dostop
Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlie the inward pacemaker current, termed If/Ih, 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 V½ was -77 ± 5.4 mV, and the reversal potential Erev was -20.5 ± 4 mV, resulting in a permeability ratio PNa/PK 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.
Sinus node inhibitors reduce the heart rate presumably by blocking the pacemaker current I f 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 IC 50 values of 0.99, 2.25, and 1.96 μM for cilobradine, ivabradine, and zatebradine, respectively. Native I f , recorded from mouse sinoatrial node cells, was slightly more efficiently blocked by cilobradine (IC 50 value of 0.62 μM) 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 ED 50 values of 1.2, 4.7, and 1.8 mg/kg for cilobradine, ivabradine, and zatebradine, respectively. β-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.
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 (IP3RI)‐associated protein (IRAG), which decreases hormone‐induced IP3‐dependent Ca2+ 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–IP3RI interaction and resulted in hypomorphic IRAGΔ12/Δ12 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 IRAGΔ12/Δ12 mice were not relaxed by cGMP, while cAMP‐mediated relaxation was unperturbed. Norepinephrine‐induced increases in Ca2+i were not decreased by cGMP in aortic smooth muscle cells from IRAGΔ12/Δ12 mice. In contrast, cGMP‐induced relaxation of potassium‐induced smooth muscle contraction was not abolished in IRAGΔ12/Δ12 mice. We conclude that cGMP‐dependent relaxation of hormone receptor‐triggered smooth muscle contraction essentially depends on the interaction of cGKI–IRAG with IP3RI.
Hyperpolarization‐activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding ...hyperpolarization‐activated, cyclic nucleotide‐gated cation channels (HCN1–4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1–4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1–4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue‐specific expression of HCN channel genes.
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 ...(τact), 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 τact 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 τact 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 τact. 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.
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