P2730 Thrombin-induced slowly inactivating sodium current by protease-activated receptor PAR1 activation in human cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BMBF Background Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) provide an opportunity to study human cardiac physiology and pathophysiology or to use for cardiac repair as well as for cardiovascular drug testing. Suitability for these purposes requires generating hiPSC cardiomyocytes that share typical electrophysiological properties of adult human cardiomyocytes. In the human heart the Na+/K+-ATPase pump current plays a major role in the regulation of contractile force and electrical stability. So far there are no data about Na+/K+-ATPase pump current function in hiPSC cardiomyocytes available. Purpose We compared the properties of Na+/K+-ATPase pump current in hiPSC cardiomyocytes from conventional monolayers (ML) culture to three-dimensional engineered heart tissue (EHT). Methods HiPSC cardiomyocytes differentiated from in-house control hiPSC cell line C25 were dissociated from ML and EHT culture. Na+/K+-ATPase pump current was recorded by whole-cell patch clamp technique at 37°C. The holding potential was -40 mV to inactivate sodium current. Current was measured in the absence of K+ and after adding 5.4 mM potassium chloride (KCL). Na+/K+-ATPase pump current was defined as the ouabain (10 µM) sensitive current. Voltage-dependency of Na+/K+-ATPase pump was determined using rectangular voltage pulses (increasing from -120 mV to +60 mV). Results Outwardly directed Na+/K+-ATPase pump could be recorded at -40 mV when KCL was added to the bath solution (5.4 mM). Currents were larger in EHT than in ML (0.8 ± 0.08 pA/pF n = 16 ML vs. 1.29 ± 0.13 pA/pF n = 28 EHT; p < 0.05). The K+-induced outward current was abolished by ouabain. The K+- and ouabain-sensitive current densities were similar in size (0.84 ± 0.11 pA/pF n = 16 ML vs. 1.12 ± 0.11 pA/pF n = 28 EHT for ouabain), indicating the measured K+-induced current was Na+/K+-ATPase pump current. Increasing extracellular K+-concentration in a stepwise manner (0,25 mM, 0,5 mM, 1 mM, 2 mM, 5,4 mM and 10 mM) showed a concentration-dependent relationship to Na+/K+-ATPase pump current with throughout higher current densities in EHT compared to ML (sensitivity to K+ not different). Na+/K+-ATPase pump current showed expected voltage-dependency with +0.23 ± 0.13 pA/pF at -120 mV and +0.89 ± 0.16 pA/pF at +60 mV (n= 18) in ML and with +0.71 ± 0.17 pA/pF at -120 mV and +1.2 ± 0.2 pA/pF at +60 mV (n= 24) in EHT. Conclusion HiPSC cardiomyocytes possess Na+/K+-ATPase pump current. Current density is in the range of human cardiomyocytes in EHT but substantially smaller in ML. 3D culturing may be needed to develop the physiological properties of Na+/K+-ATPase pump current.

Download Full-text

CaMKII delta interaction with neuronal sodium channel Nav1.8 contributes to arrhythmogenic triggers in failing human and mouse cardiomyocytes

European Heart Journal ◽

10.1093/ehjci/ehaa946.3693 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

N Dybkova ◽

S Ahmad ◽

P Tirilomis ◽

P Bengel ◽

S Pabel ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Current ◽

Subcellular Localisation ◽

Dependent Protein Kinase ◽

Human Cardiomyocytes ◽

Whole Cell Patch Clamp ◽

Relevant Role ◽

Dependent Protein ◽

First Time ◽

Human And Mouse

Abstract In heart failure, enhanced persistent current through neuronal sodium channel NaV1.8 (INaL) may induce influx of Na+ into cardiomyocytes. This may cause Ca2+ influx via the Na+/Ca2+ exchanger leading to increased proarrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ leak. This Ca2+ may activate Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) which can induce INaL augmentation by phosphorylating NaV1.5 channels leading to a vicious cycle between INaL and CaMKIIδ. Here, we examined whether CaMKIIδ associates with NaV1.8 in human and mouse cardiomyocytes thereby regulating its function. Interaction and co-localisation of CaMKIIδ and NaV1.8 were confirmed by co-immunoprecipitation and immunocytochemistry. Whole-cell patch clamp showed a potent reduction of INaL after addition of novel specific Nav1.8 blockers, either A-803467 (30 nmol/L) or PF-01247324 (1 μmol/L) in failing mouse cardiomyocytes overexpressing CaMKIIδc (CaMKIIδc+/T: −109.4±10.6 vs A-803467: −56.9±11.7 and PF-01247324:−-69.9±8.6 A*ms*F-1). In failing human cardiomyocytes inhibition of either NaV1.8 or CaMKIIδ using AIP (1 μmol/L) or AIP and PF-01247324 together led to a significant and comparable decrease of INaL (control: −93.7±7.1 vs PF-01247324: −56.8±6.6; AIP: −44.2±6.6; AIP+PF-01247324: −39.8±5.4 A*ms*F-1). Furthermore, to confirm whether observed alterations in INaL after inhibition of NaV1.8 are not due to an overall reduction in peak sodium current (INa) we measured INa properties in mouse cardiomyocytes. Importantly, we observed no difference neither in the peak nor in inactivation between wild type (WT), WT with PF-01247324 and in mice lacking NaV1.8. Using confocal microscopy we investigated whether inhibition of the NaV1.8-mediated INaL could attenuate the increase of proarrhythmogenic SR Ca2+ spark frequency (CaSpF) caused by overexpression of CaMKIIδ in mice. We observed a significant reduction of CaSpF in both NaV1.8 inhibitor groups (PF-01247324: 0.51±0.08 and A-803467: 0.57±0.08 μm–1 s–1) compared to control (1.00±0.13 μm–1 s–1). Incubation of human failing cardiomyocytes with either AIP (0.35±0.06 μm–1 s–1) or PF-01247324 (0.44±0.11 μm–1 s–1), or blocking CaMKIIδ and NaV1.8 together (0.30±0.08 μm–1 s–1) resulted in significant decrease of CaSpF compared to control (0.89±0.13 μm–1 s–1). In conclusion, we show for the first time subcellular localisation of the neuronal sodium channel NaV1.8 and its interaction with CaMKIIδ in both human and mouse ventricular cardiomyocytes. Moreover, pharmacological inhibition of NaV1.8 caused a reduction of the augmented INaL and spontaneous diastolic SR-Ca2+ release in both failing human and mouse cardiomyocytes. NaV1.8 and CaMKIIδ interaction seem to play a relevant role for the generation of arrhythmogenic triggers (INaL & spontaneous diastolic SR-Ca2+ release) in both human and mouse cardiomyocytes from failing hearts. Funding Acknowledgement Type of funding source: None

Download Full-text

Effects of Amiodarone and Dronedarone on Voltage‐Dependent Sodium Current in Human Cardiomyocytes

Journal of Cardiovascular Electrophysiology ◽

10.1046/j.1540-8167.2003.03064.x ◽

2003 ◽

Vol 14 (8) ◽

pp. 885-890 ◽

Cited By ~ 74

Author(s):

Nathalie Lalevée ◽

Stéphanie Barrère‐lemaire ◽

Patrick Gautier ◽

Joël Nargeot ◽

Sylvain Richard

Keyword(s):

Sodium Current ◽

Human Cardiomyocytes ◽

Voltage Dependent

Download Full-text

Causal SNP regulating FAM13B expression identified for the Chr. 5q31 atrial fibrillation susceptibility locus

10.1101/719914 ◽

2019 ◽

Author(s):

Gregory Tchou ◽

Shamone Gore-Panter ◽

Jeffrey Hsu ◽

Fang Liu ◽

Nana Liu ◽

...

Keyword(s):

Gene Expression ◽

Atrial Fibrillation ◽

Plasma Membrane ◽

Genetic Variant ◽

Risk Allele ◽

Sodium Current ◽

Susceptibility Locus ◽

Enhancer Activity ◽

Human Cardiomyocytes ◽

The Common

AbstractRationaleOur prior RNA sequencing study found that FAM13B gene expression in human left atrial appendages was strongly associated with an atrial fibrillation (AF) susceptibility-associated variant on chr. 5q31.ObjectiveTo identify the common genetic variant responsible for regulating FAM13B expression and the effect of FAM13B expression on cardiomyocyte gene expression in order to gain insight into the functional mechanism of the chr. 5q31 AF susceptibility locus.Methods and ResultsBy taking advantage of a smaller linkage disequilibrium block in African descent subjects and available chromatin conformation data, we identified the common single nucleotide polymorphism (SNP) rs17171731 as a candidate genetic variant controlling FAM13B gene expression in the left atrium. Functional analysis demonstrated that the AF risk allele of rs17171731 had less enhancer activity than the protective allele. Gel mobility shift studies determined that the risk allele bound to an additional protein that may function as a transcriptional repressor. Knockdown of FAM13B expression in stem cell-derived human cardiomyocytes (iCM) altered the expression of >1000 genes and modified the sodium current, consistent with increased susceptibility to atrial fibrillation. Transfection of GFP tagged FAM13B into iCMs demonstrated expression on the plasma membrane and at the Z-disk.ConclusionsThe chr. 5q31 AF risk variant was identified as rs17171731, with the risk allele having less enhancer activity, leading to decreased expression of FAM13B, which resides on the plasma membrane and the Z-disk, and appears to play a role in the regulation of cardiomyocyte gene expression and the late sodium current.

Download Full-text