Erich Wettwer

The G Protein–Gated Potassium Current IK,ACh Is Constitutively Active in Patients With Chronic Atrial Fibrillation

Background— The molecular mechanism of increased background inward rectifier current (IK1) in atrial fibrillation (AF) is not fully understood. We tested whether constitutively active acetylcholine (ACh)-activated IK,ACh contributes to enhanced basal conductance in chronic AF (cAF). Methods and Results— Whole-cell and single-channel currents were measured with standard voltage-clamp techniques in atrial myocytes from patients with sinus rhythm (SR) and cAF. The selective IK,ACh blocker tertiapin was used for inhibition of IK,ACh. Whole-cell basal current was larger in cAF than in SR, whereas carbachol (CCh)-activated IK,ACh was lower in cAF than in SR. Tertiapin (0.1 to 100 nmol/L) reduced IK,ACh in a concentration-dependent manner with greater potency in cAF than in SR (−logIC50: 9.1 versus 8.2; P<0.05). Basal current contained a tertiapin-sensitive component that was larger in cAF than in SR (tertiapin [10 nmol/L]-sensitive current at −100 mV: cAF, −6.7±1.2 pA/pF, n=16/5 [myocytes/patients] versus SR, −1.7±0.5 pA/pF, n=24/8), suggesting contribution of constitutively active IK,ACh to basal current. In single-channel recordings, constitutively active IK,ACh was prominent in cAF but not in SR (channel open probability: cAF, 5.4±0.7%, n=19/9 versus SR, 0.1±0.05%, n=16/9; P<0.05). Moreover, IK1 channel open probability was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05) without changes in other channel characteristics. Conclusions— Our results demonstrate that larger basal inward rectifier K+ current in cAF consists of increased IK1 activity and constitutively active IK,ACh. Blockade of IK,ACh may represent a new therapeutic target in AF.

Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin.

In various mammalian species, shapes of action potentials vary within the cardiac wall because of differences in transient outward current (Ito). A prominent Ito exists in human ventricular myocytes, but cells have not been separated according to their original localization. Human ventricular myocytes were isolated from separated subepicardial and subendocardial tissue, and regional variations in Ito were studied. Ito was larger in subepicardial than subendocardial cells. Current density at +60 mV was 7.9 +/- 0.7 pA/pF (n = 28) in subepicardial cells and 2.3 +/- 0.3 pA/pF (n = 16) in subendocardial cells. When cells from explanted failing and nonfailing donor hearts were compared, Ito was not different in subepicardial cells; however, it was larger in subendocardial cells from nonfailing hearts. The potential of half-maximal activation (V0.5) was more positive in subendocardial cells (+25.6 +/- 3.5 mV, n = 15) than in subepicardial cells (+9.2 +/- 1.8 mV, n = 28). There was no difference in V0.5 between cells from failing and nonfailing hearts. Ito inactivation was similar in all cell types and independent of membrane depolarization (time constant [tau] = approximately 60 milliseconds at 22 degrees C). The potential of half-maximal steady-state inactivation was similar in all cell types. Recovery from inactivation of Ito was fast in subepicardial cells at -100 mV (tau = 24 +/- 4 milliseconds, n = 6), exceeding control values transiently (overshoot), and slow at -40 mV without overshoot (tau = 638 +/- 91 milliseconds, n = 6). In subendocardial cells, Ito recovered at -100 mV with a fast phase (tau = 25 milliseconds) and a slow phase (tau = 328 milliseconds), and recovery was not complete after 6 seconds at -100 mV. In conclusion, regional differences in Ito between subepicardial and subendocardial cells may have clinical implications with respect to rhythmic disturbance during heart failure.

L-type Ca2+ current downregulation in chronic human atrial fibrillation is associated with increased activity of protein phosphatases.

Background—Although downregulation of L-type Ca2+ current (ICa,L) in chronic atrial fibrillation (AF) is an important determinant of electrical remodeling, the molecular mechanisms are not fully understood. Here, we tested whether reduced ICa,L in AF is associated with alterations in phosphorylation-dependent channel regulation. Methods and Results—We used whole-cell voltage-clamp technique and biochemical assays to study regulation and expression of ICa,L in myocytes and atrial tissue from 148 patients with sinus rhythm (SR) and chronic AF. Basal ICa,L at +10 mV was smaller in AF than in SR (−3.8±0.3 pA/pF, n=138/37 [myocytes/patients] and −7.6±0.4 pA/pF, n=276/86, respectively; P<0.001), though protein levels of the pore-forming &agr;1c and regulatory &bgr;2a channel subunits were not different. In both groups, norepinephrine (0.01 to 10 &mgr;mol/L) increased ICa,L with a similar maximum effect and comparable potency. Selective blockers of kinases revealed that basal ICa,L was enhanced by Ca2+/calmodulin-dependent protein kinase II in SR but not in AF. Norepinephrine-activated ICa,L was larger with protein kinase C block in SR only, suggesting decreased channel phosphorylation in AF. The type 1 and type 2A phosphatase inhibitor okadaic acid increased basal ICa,L more effectively in AF than in SR, which was compatible with increased type 2A phosphatase but not type 1 phosphatase protein expression and higher phosphatase activity in AF. Conclusions—In AF, increased protein phosphatase activity contributes to impaired basal ICa,L. We propose that protein phosphatases may be potential therapeutic targets for AF treatment.

Role of IKur in Controlling Action Potential Shape and Contractility in the Human Atrium: Influence of Chronic Atrial Fibrillation

Background—The ultrarapid outward current IKur is a major repolarizing current in human atrium and a potential target for treating atrial arrhythmias. The effects of selective block of IKur 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 &mgr;mol/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 &mgr;mol/L). Computer simulations of IKur block in human atrial APs predicted secondary increases in ICa,L and in the outward rectifiers IKr and IKs, with smaller changes in AF than SR. The indirect increase in ICa,L was supported by a positive inotropic effect of 4-aminopyridine without direct effects on ICa,L in atrial but not ventricular preparations. In accordance with the model predictions, block of IKr with E-4031 converted APD shortening effects of IKur block in SR into AP prolongation. Conclusions—Whether inhibition of IKur prolongs or shortens APD depends on the disease status of the atria and is determined by the level of electrical remodeling.

Differences between outward currents of human atrial and subepicardial ventricular myocytes.

1. Outward currents were studied in myocytes isolated from human atrial and subepicardial ventricular myocardium using the whole‐cell voltage clamp technique at 22 degrees C. The Na+ current was inactivated with prepulses to ‐40 mV and the Ca2+ current was eliminated by both reducing extracellular [Ca2+] to 0.5 mM and addition of 100 microM CdCl2 to the bath solution. 2. In human myocytes, three different outward currents were observed. A slowly inactivating sustained outward current, I(so), was found in atrial but not ventricular myocytes. A rapidly inactivating outward current, I(to), of similar current density was observed in cells from the two tissues. An additional uncharacterized non‐inactivating background current of similar size was observed in atrial and in ventricular myocytes. 3. I(to) and I(so) could be differentiated in atrial myocytes by their different kinetics and potential dependence of inactivation, and their different sensitivities to block by 4‐amino‐pyridine, suggesting that two individual channel types were involved. 4. In atrial cells, inactivation of I(to) was more rapid and steady‐state inactivation occurred at more negative membrane potentials than in ventricular cells. Furthermore, the recovery of I(to) from inactivation was slower and without overshoot in atrial myocytes. In addition, 4‐aminopyridine‐induced block of I(to) was more efficient in atrial than in ventricular cells. These observations suggest that the channels responsible for atrial and ventricular I(to) were not identical. 5. We conclude that the differences in outward currents substantially contribute to the particular shapes of human atrial and ventricular action potentials. The existence of I(so) in atrial cells only provides a clinically interesting target for anti‐arrhythmic drug action, since blockers of I(so) would selectively prolong the atrial refractory period, leaving ventricular refractoriness unaltered.

Electrophysiological properties of human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) have gained considerable interest due to their potential use for cell replacement therapy and tissue engineering. One strategy is to differentiate these bone marrow stem cells in vitro into cardiomyocytes prior to implantation. In this context ion channels can be important functional markers of cardiac differentiation. At present there is little information about the electrophysiological behaviour of the undifferentiated hMSC. We therefore investigated mRNA expression of 26 ion channel subunits using semiquantitative RT‐PCR and recorded transmembrane ion currents with the whole‐cell voltage clamp technique. Bone marrow hMSC were obtained from healthy donors. The cells revealed a distinct pattern of ion channel mRNA with high expression levels for some channel subunits (e.g. Kv4.2, Kv4.3, MaxiK, HCN2, and α1C of the L‐type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly activated at potentials positive to +20 mV. This current was identified as a large‐conductance voltage‐ and Ca2+‐activated K+ current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC50= 340 μm) and its inhibition by 100 nm iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to –30 mV. This current was selectively inhibited by clofilium (IC50= 0.8 μm). Ba2+ inward currents, stimulated by 1 μm BayK 8644 were found in a few cells, indicating the expression of functional L‐type Ca2+ channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell function.

Human inward rectifier potassium channels in chronic and postoperative atrial fibrillation

OBJECTIVE We showed recently that the 825T allele of the G-protein beta 3-subunit C825T polymorphism is associated with large inward rectifier K(+) currents I(K1) but low acetylcholine-activated K(+) current I(K,ACh) amplitudes. During chronic atrial fibrillation (AF), I(K1) and I(K,ACh) current densities were increased when compared to sinus rhythm (SR). It is unknown whether chronic AF and G beta 3 gene status are independent contributors to atrial K(+) current activity. We measured I(K1) and I(K,ACh) in tissue from AF patients with different G beta 3 genotypes and assessed the relation between the I(K1) and I(K,ACh) amplitudes and the incidence of postoperative AF. METHODS We measured the amplitudes of I(K1) and I(K,ACh) in atrial myocytes from 26 patients with sinus rhythm (SR) and from 16 patients with chronic AF (>6 months). The K(+) currents were measured with standard patch-clamp techniques. The G beta 3 gene status of the patients was determined by PCR and restriction analysis. RESULTS At -100 mV, the amplitude of I(K1) was larger in AF (10.9+/-1.0 pA/pF, n=49/16, cells/patients) than in SR (6.3+/-0.6 pA/pF, n=68/26, P<0.05), whereas the amplitude of I(K,ACh) was smaller in chronic AF (2.9+/-0.7 pA/pF, n=49/16) than in SR (6.3+/-0.7 pA/pF, n=68/26, P<0.05). These changes were independent of the patient G beta 3 gene status. Eight patients out of 26 in the SR group (31%) developed postoperative AF. When analysed based on incidence of postoperative AF, current amplitudes did not differ significantly. CONCLUSION We provide evidence for up-regulation of I(K1) but down-regulation of I(K,ACh) in chronic AF which are independent of G beta 3 gene status. Atrial myocytes from patients who are in SR but later develop postoperative AF have no manifestation of altered I(K1) and I(K,ACh) at the time of cardiac surgery. Our results suggest that the AF-related changes of I(K1) and I(K,ACh) may be a consequence of or a contributory factor to chronic AF.

Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative β-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 (Ito) are not mimicked by coexpression of the α‐subunit Kv4.3 with its known β‐subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional β‐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 Ito‐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 Ito channel complex in human heart.

G-Protein β3-Subunit 825T Allele Is Associated With Enhanced Human Atrial Inward Rectifier Potassium Currents

BACKGROUND A C825T polymorphism was recently identified in the human gene encoding for the beta(3)-subunit of heterotrimeric G proteins. The 825T allele is associated with a splice variant of Gbeta(3) and enhanced signal transduction. We hypothesized that patients carrying the 825T allele exhibit the modified Gbeta(3) phenotype. The resulting enhancement of signal transduction should be detectable in the Gbetagamma-dimer-mediated acetylcholine-stimulated K(+) current (I(K,ACh)). METHODS AND RESULTS Seventy patients undergoing cardiac surgery were genotyped for the C825T polymorphism. In right atrial myocytes from these patients, the inward rectifier K(+) currents (I(K1), I(K,ACh)) were studied with the whole-cell patch-clamp technique. Background current I(K1) was measured with depolarizing ramp pulses and quantified as inward current at -100 mV; mean amplitudes were (pA/pF) 4.98+/-0.49 (n=30/93 patients/cells) in patients with CC genotype, 4.25+/-0.36 (n=31/121 patients/cells) with TC, and 7. 46+/-1.14 (n=9/32 patients/cells; P<0.05) with TT. Conversely, mean I(K,ACh), which is maximally activated by carbachol (2 micromol/L), was reduced in patients with TT genotype (pA/pF, 4.30+/-1.33, n=9/27 patients/cells; P<0.05) compared with the other 2 groups (6.56+/-0. 54, n=30/80 and 6.16+/-0.45, n=31/117 patients/cells, for CC and TC genotype, respectively). Essentially similar results were obtained with adenosine (1 mmol/L). CONCLUSIONS We found an association between the Gbeta(3) 825T allele and amplitude of human atrial I(K1) and I(K,ACh). Increased background current density in TT carriers could shorten action potential duration and may be due to I(K,ACh) being constitutively active in this genotype.

Adult zebrafish heart as a model for human heart? An electrophysiological study.

The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K(+) channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na(+) channels, L-type Ca(2+) channels contribute to the plateau phase and I(Kr) channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca(2+) current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca(2+) channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype.