Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes

Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (IK1). Here, IK1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. IK1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. IK1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and IK,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that IK1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs

Ionic mechanisms limiting cardiac repolarization-reserve in humans compared to dogs.

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50–100 nmol l−1 dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l−1 BaCl2) and IKs block (1 μmol l−1 HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ∼30% larger and ∼29% smaller in human, and Na+–Ca2+ exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKsα-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human–canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease

The transmembrane \u3b2-subunits KCNE1, KCNE2, and DPP6 modify pharmacological effects of the antiarrhythmic agent tedisamil on the transient outward current i to

Accessory beta-subunits modulate the pharmacology of ion channel blockers. The aim was to investigate differences in effects of the antiarrhythmic agent and open-channel blocker tedisamil on transient outward current I(to) (Kv4.3) when coexpressed with beta-subunits potassium voltage-gated channel, Isk-related family, member 1 (KCNE1), potassium voltage-gated channel, Isk-related family, member 2 (KCNE2), or dipeptidyl-aminopeptidase-like protein 6 (DPP6) which modulate I(to) kinetics. Tedisamil inhibited I(to) with IC(50) values of 16 microM for Kv4.3+KChIP2, 11 microM in the presence of KCNE1, and 14 microM for KCNE2. Values were higher in the presence of DPP6 or DPP6+KCNE2 (35 and 26 microM). K(d) values of tedisamil binding and rate constants were not affected by KCNE or DPP6. I(to) kinetics were accelerated by KCNE and DPP6, inactivation to a larger extent with DPP6. Tedisamil did not affect activation time course but apparently accelerated inactivation in all channel subunit combinations tested. Deletion of the intracellular domain of KCNE2 or DPP6 resulted in slowing of kinetics and increased tedisamil sensitivity (IC(50) 4 and 7 microM). It is concluded that apparent effects of DPP6 and deletion mutants (KCNE2 and DPP6) are due to the acceleration or slowing effects of the beta-subunits on I(to) kinetics

Tissue Slices from Adult Mammalian Hearts as a Model for Pharmacological Drug Testing

Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 μm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD90 with a half maximal effective concentration (EC50) of 4.5 μM. The IKr blocker E4031 and neuroleptic drug risperidone prolonged APD90 with EC50 values of 31 nM and 0.67 μM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m ⋅ s-1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Impaired glycosylation blocks DPP10 cell surface expression and alters the electrophysiology of i to channel complex

DPP10 is a transmembrane glycosylated protein belonging to the family of dipeptidyl aminopeptidase-like proteins (DPPLs). DPPLs are auxiliary subunits involved in the regulation of voltage-gated Kv4 channels, key determinants of cardiac and neuronal excitability. Although it is known that DPPLs are needed to generate native-like currents in heterologous expression systems, the molecular basis of this involvement are still poorly defined. In this study, we investigated the functional relevance of DPP10 glycosylation in modulating Kv4.3 channel activities. Using transfected Chinese hamster ovary (CHO) cells to reconstitute Kv4 complex, we show that the pharmacological inhibition of DPP10 glycosylation by tunicamycin and neuraminidase affects transient outward potassium current (I (to)) kinetics. Tunicamycin completely blocked DPP10 glycosylation and reduced DPP10 cell surface expression. The accelerating effects of DPP10 on Kv4.3 current kinetics, i.e. on inactivation and recovery from inactivation, were abolished. Neuraminidase produced different effects on current kinetics than tunicamycin, i.e., shifted the voltage dependence to more negative potentials. The effects of tunicamycin on the native I (to) currents of human atrial myocytes expressing DPP10 were similar to those of the KV4.3/KChIP2/DPP10 complex in CHO cells. Our results suggest that N-linked glycosylation of DPP10 plays an important role in modulating Kv4 channel activities

N-glycosylation of the mammalian dipeptidyl aminopeptidase-like protein 10 (DPP10) regulates trafficking and interaction with Kv4 channels

The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels

Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes

1. Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (I(to)) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, I(to) was analysed after appropriate voltage inactivation of sodium current and block of calcium current. 2. Tedisamil reduced the amplitude of peak I(to), but did not affect the amplitude of non-inactivating outward current. The drug accelerated the apparent rate of I(to) inactivation. The reduction in time constant of I(to) inactivation depended on drug concentration, the apparent IC(50) value was 4.4 μM. 3. Tedisamil affected I(to) amplitude in a use-dependent manner. After 2 min at −80 mV, maximum block of I(to) was reached after 4–5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency-dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1±1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81±40 ms and 51±8 ms, respectively, n.s.). 4. From the onset of fractional block of I(to) by tedisamil during the initial 60 ms of a clamp step, we calculated k(1)=9×10(6) mol(−1) s(−1) for the association rate constant, and k(2)=23 s(−1) for the dissociation rate constant. The resulting apparent K(D) was 2.6 μM and is similar to the IC(50) value. 5. The effects of tedisamil on I(to) could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of I(to) and that this effect probably contributes to the antiarrhythmic potential of this drug