Torsten Christ

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.

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.

Human Atrial Ion Channel and Transporter Subunit Gene-Expression Remodeling Associated With Valvular Heart Disease and Atrial Fibrillation

Background—Valvular heart disease (VHD), which often leads to atrial fibrillation (AF), and AF both cause ion-channel remodeling. We evaluated the ion-channel gene expression profile of VHD patients, in permanent AF (AF-VHD) or in sinus rhythm (SR-VHD), in comparison with patients without AF or VHD, respectively. Methods and Results—We used microarrays containing probes for human ion-channel and Ca2+-regulator genes to quantify mRNA expression in atrial tissues from 7 SR-VHD patients and 11 AF-VHD patients relative to 11 control patients in SR without structural heart disease (SR-CAD). From the data set, we selected for detailed analysis 59 transcripts expressed in the human heart. SR-VHD patients differentially expressed 24/59 ion-channel and Ca2+-regulator transcripts. There was significant overlap between VHD groups, with 66% of genes altered in SR-VHD patients being similarly modified in AF-VHD. Statistical differences between the AF- and SR-VHD groups identified the specific molecular portrait of AF, which involved 12 genes that were further confirmed by real-time reverse transcription–polymerase chain reaction. For example, phospholamban, the &bgr;-subunit MinK (KCNE1) and MIRP2 (KCNE3), and the 2-pore potassium channel TWIK-1 were upregulated in AF-VHD compared with SR-VHD, whereas the T-type calcium-channel Cav3.1 and the transient-outward potassium channel Kv4.3 were downregulated. Two-way hierarchical clustering separated SR-VHD from AF-VHD patients. AF-related changes in L-type Ca2+-current and inward-rectifier current were confirmed at protein and functional levels. Finally, for 13 selected genes, SR restoration reversed ion-channel remodeling. Conclusions—VHD extensively remodels cardiac ion-channel and transporter expression, and AF alters ion-channel expression in VHD patients.

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.

Adipocyte Fatty Acid-Binding Protein Suppresses Cardiomyocyte Contraction. A New Link Between Obesity and Heart Disease

Rationale: Adipocyte fatty acid–binding protein (FABP4) is a member of the intracellular lipid-binding protein family and is predominantly expressed in adipose tissue. Emerging evidence suggests that FABP4 plays a role in some aspects of the metabolic syndrome including the development of type 2 diabetes and atherosclerosis. We have recently reported that secretory products from human adipocytes directly and acutely depressed cardiac contractile function. Objective: The purpose of this study was to identify this adipocyte-derived cardiodepressant factor. Methods and Results: Through mass spectrometry and immunoblotting, we have identified this cardiodepressant factor as FABP4. FABP4 represents 1.8% to 8.1% of total protein secreted by adipocytes in extracellular medium. FABP4 acutely depressed shortening amplitude as well as intracellular systolic peak Ca2+ in a dose-dependent manner in isolated rat cardiomyocytes. Heart-specific FABP isoform (FABP3) revealed a similar cardiodepressant effect. The N-terminal amino acids 1 to 20 of FABP4 could be identified as the most effective cardiodepressive domain. We could exclude any effect of FABP4 on action potential duration and L-type Ca2+ current, suggesting a reduced excitation-contraction gain caused by FABP4 as the main inhibitory mechanism. Conclusion: We conclude that the release of FABP4 from adipocytes may be involved in the development of cardiac contractile dysfunction of obese subjects.

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.

Pathology-specific effects of the IKur/Ito/IK,ACh blocker AVE0118 on ion channels in human chronic atrial fibrillation

This study was designed to establish the pathology‐specific inhibitory effects of the IKur/Ito/IK,ACh blocker AVE0118 on atrium‐selective channels and its corresponding effects on action potential shape and effective refractory period in patients with chronic AF (cAF).

Transmural expression of ion channels and transporters in human nondiseased and end-stage failing hearts

The cardiac action potential is primarily shaped by the orchestrated function of several different types of ion channels and transporters. One of the regional differences believed to play a major role in the progression and stability of the action potential is the transmural gradient of electrical activity across the ventricular wall. An altered balance in the ionic currents across the free wall is assumed to be a substrate for arrhythmia. A large fraction of patients with heart failure experience ventricular arrhythmia. However, the underlying substrate of these functional changes is not well-established as expression analyses of human heart failure (HF) are sparse. We have investigated steady-state RNA levels by quantitative polymerase chain reaction of ion channels, transporters, connexin 43, and miR-1 in 11 end-stage HF and seven nonfailing (NF) hearts. The quantifications were performed on endo-, mid-, and epicardium of left ventricle, enabling us to establish changes in the transmural expression gradient. Transcripts encoding Cav1.2, HCN2, Kir2.1, KCNE1, SUR1, and NCX1 were upregulated in HF compared to NF while a downregulation was observed for KChIP2, SERCA2, and miR-1. Additionally, the transmural gradient of KCNE1, KChIP2, Kir6.2, SUR1, Nav1.5, NCX1, and RyR2 found in NF was only preserved for KChiP2 and Nav1.5 in HF. The transmural gradients of NCX1, Nav1.5, and KChIP2 and the downregulation of KChIP2 were confirmed by Western blotting. In conclusion, our results reveal altered expression of several cardiac ion channels and transporters which may in part explain the increased susceptibility to arrhythmia in end-state failing hearts.

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells.

Previously, mouse bone marrow-derived stem cells (MSC) treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1–3 passages, cultures were exposed for 24 h to 5-azacytidine (3 μM) followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca2+ currents. Almost all cells showed outwardly rectifying K+ currents of rapid or slow activation kinetics. Mean current amplitude at +60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks. Expression levels of mRNAs for the K+ channels Kv1.1, Kv1.5, Kv2.1, Kv4.3 and KCNMA1 and for the Ca2+ channel Cav1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K+ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation of hMSC into cardiomyocytes, treatment with 5-azacytidine caused profound changes in current density.