The most common arrhythmia observed in the clinic is atrial fibrillation (AF). As such, the identification of the cellular mechanisms of AF and development of new safe and effective antiarrhythmic drugs are a priority(1). However, the physiology of human atrial cardiomyocytes may not be replicated with preclinical studies using non-cardiac cells and non-human animal models and they may not adequately predict patient efficacy and safety(2).
Results from studies carried out to validate human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-ACMs) generated by Axol Bioscience are outlined here. First characterized at the molecular level using immunocytochemistry with atrial specific markers, the atrial phenotype of Axol hiPSC-ACMs then went through functional validation using manual patch clamp recordings of action potential (AP) parameters.
Modulators of the atrial specific acetylcholine-activated inward-rectifying potassium current (IKACh) and the ultrarapid delayed rectifier potassium current (Ikur) further confirmed the atrial phenotype(1).
Materials and Methods
Cell Culture: In accordance with the manufacturer’s instructions, Human iPSC-derived atrial cardiomyocytes (Axol Bioscience Ltd.) were generated through a variation (retinoid acid) of the Burridge et al method(3), seeded and cultured.
Immunocytochemistry: Cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 and blocked with 5% donkey serum. Primary antibody was incubated overnight 4 ̊C, and secondary antibody coupled to Alexa Fluor® dyes (Invitrogen) applied for 2 hours.
Manual Patch Clamp (MPC): 7-10 days after cell seeding AP were recorded from Axol hiPSC-ACM. Perforated patch (100 μg/ml gramicidin) was used to take records at room temperature in current clamp mode. EPC10 amplifiers and PatchMaster software (HEKA Elektronik, Germany) were used to acquire data. Digitisation at 20 kHz occurred after analog signals were low-pass filtered at 10 kHz. CAPA software (SSCE UG, Germany) was used to analyze pontaneous AP. AP parameters: maximum diastolic potential (MDP), upstroke velocity (dV/dtmax), AP amplitude (APA), AP duration at 20, 50 and 90% repolarization (APD20, APD50, APD90), and frequency (Freq). Data are presented as mean ± SEM. Paired student’s t-test of control values compared to the effect of compound application was used to determine significance. * P<0.05, ** P<0.01, *** P<0.001.
1. Expression of Cardiac Markers in Axol hiPSC-ACM
Axol hiPSC-ACM express protein makers characteristic of atrial cardiomyocytes
Figure 1: Cardiac protein marker expression was detected in Axol hiPSC-ACM by immunocytochemistry
The expression of two atrial-specific markers (atrial myosin light chain MLC2a and atrial natriuretic peptide ANP), one ventricular marker (ventricular myosin light chain; MLC2v), and one general cardiac marker (Troponin T; TropT) were evaluated in hiPSC-ACM. (A) Higher expression levels of MLC2a (red) were detected compared to MLC2v (green). (B) Cells positive for MLC2a (red) also expressed ANP (green). (C) Expression of Troponin T (red) was also confirmed in ANP (green) positive cells. Image Credit: Axol Bioscience
2. Action Potential Characteristics
Axol hiPSC-ACM elicit spontaneous action potentials and are suitable for pacing experiments
Figure 2: Characteristics of spontaneous and evoked action potentials from Axol hiPSC-ACM
Representative traces of spontaneous (A) and evoked (1 Hz; B) AP recorded under control conditions. (C) Average AP parameters. Image Credit: Axol Bioscience
3. Functional Expression of Core Cardiac Ion Channel Pharmacology
Pharmacological modulation of spontaneous AP confirmed functional expression of core cardiac currents
Figure 3: Confirmed functional expression of core cardiac currents in Axol hiPSC-ACM
The presence of three core cardiac currents in Axol hiPSC-ACM action potentials were confirmed using Lidocaine (INa), Nifedipine (ICa,L), and E-4031 (IKr). Representative spontaneous AP under control conditions (grey) and in the presence of 100 μM Lidocaine (A, green), 100 nM Nifedipine (B, blue), and 100 nM E-4031 (C, red). Early after depolarisations (EADs), indicative of arrhythmic events, were observed following E-4031 application (C, arrow). D: Average effect (% of control) on AP parameters, N ≥ 4. Image Credit: Axol Bioscience
4. Atrial-specific Ion Channel Pharmacology
Pharmacological modulation of spontaneous AP confirmed expression of functional atrial phenotype
Figure 4: Functional confirmation of the atrial phenotype of Axol iPSC-ACM
The atrial phenotype of Axol iPSC-ACM was confirmed using compounds (4-AP for IKur and Carbachol for IKACh) which modulate atrial specific currents that are also targets for AF. Representative spontaneous AP in control conditions (grey) and the presence of 50 μM 4-AP (A, blue) and 1 μM Carbachol (B, orange). Concordant bar graphs show the average effect (% of control) on AP parameters, N ≥ 5. Image Credit: Axol Bioscience
- An atrial-like phenotype suitable for use on electrophysiological platforms is expressed with Axol hiPSC-ACMs as confirmed by the molecular and pharmacological data presented here.
- In the development of improved translational models of AF, Axol hiPSC-ACMs represent a promising tool as:
- Axol iPSC-ACMs express atrial-specific markers
- Sensitivity to a core panel of cardiac ion channel inhibitors are shown with spontaneous APs
- Selective modulators of atrial-specific currents such as IKACh and IKur (validated targets of AF drug discovery) were used to confirm an atrial phenotype.
Produced from materials originally authored by Saïd El-Haou, Sarah Williams, Louise Webdale, Robert W. Kirby, Kathy Sutton, Nicola Dark, Mariagrazia Paonessa and Marc Rogers from Metrion Biosciences and Axol Bioscience.
References and Further Reading
- El-Haou S et al. (2015). J Cardiovasc Pharmacol. 66 (5); 412-31
- Devalla et al. (2015). EMBO Mol Med. 7(4); 394-410
- Burridge et al. (2014). Nat Methods. 11(8): 855-60
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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