Atrial fibrillation is the most common arrhythmia observed in the clinic, affecting 6 million patients in Europe and accounting for 30% of strokes. The prevalence of atrial fibrillation continues to grow due to an aging population and is expected to double over the next 50 years. Given the need to develop safer and more effective anti-arrhythmic therapeutics, considerable efforts have been made to understand the cellular mechanisms of the disease and translate this knowledge into innovative treatments.
Traditional strategies for drug discovery in the area of atrial fibrillation are based on preclinical animal and non-cardiac cell models. Sadly, these often fail to reliably predict treatment efficacy and safety in patients, as evidenced by the lack of new drugs licensed in recent years. Human atrial cardiomyocyte primary cells are a more promising alternative, however the viability and yield of live cells from human hearts is low, and studies can only be performed on a limited amount of material. A further factor limiting the usefulness of primary cells is the challenge of maintaining the cells in culture for long periods. Here, we discuss the benefits of using iPSC-derived atrial cardiomyocytes to overcome some of these challenges (you can also find out more by downloading our latest whitepaper on the topic).
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iPSC-derived atrial cardiomyocytes: a better model for atrial fibrillation research
Human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes are an ideal platform for modelling atrial fibrillation for two key reasons. First, they are human atrial cells, so they are more relevant than animal models and other human cell types. Second, they provide a sustained and plentiful source of cells to work with (overcoming the limitations imposed by using primary cells).
Despite their benefits, human iPSC cells can be challenging to produce. To enable their use as a physiologically relevant tool to support translational atrial fibrillation research, manufacturing processes must be capable of delivering a consistent and scalable supply.
To address this issue, our team has developed a line of iPSC-derived atrial cardiomyocytes that can be used to support drug discovery in this area. The cells were derived from iPSCs using footprint-free episomal reprogramming methods, via the addition of specific differentiation factors at critical time points to drive atrial fate.
Immunocytochemical analysis of the atrial cardiomyocytes generated reveals strong expression of cardiac and atrial-specific proteins. Figure 1 shows that the cells are richly labelled by antibodies directed against Troponin T and myosin light chain (MLC), confirming they are cardiac myocytes expressing contractile proteins associated with muscle sarcomeres. Their atrial differentiation is indicated by enrichment for atrial MLC (over ventricular MLC), and strong labelling for atrial natriuretic peptide (ANP) which is specifically secreted by atrial myocytes upon cell stretching.
Figure 1: Visualisation of cardiac and atrial-specific protein markers in hiPSC-atrial cardiomyocytes by immunocytochemistry.
Genotypic characterisation of iPSC-derived atrial cardiomyocytes
For atrial cardiomyocytes to be useful as translationally predictive and reliable models for disease research, consistency and reproducibility are essential. The atrial cardiomyocyte genotype of Axol’s Human iPSC-derived Atrial Cardiomyocytes was characterised using gene chip microarray analysis of cardiac and atrial-specific markers (Figure 2).
Figure 2: Gene chip microarray expression for A) genes that signify pluripotency compared to iPS cells B) cardiac genes (red), atrial-specific genes (dark blue), cardiac ion channel genes (light blue), and ventricular-specific genes (yellow), compared to signals detected in iPSC-derived ventricular cardiomyocytes.
Key iPSC markers SOX2, KLF4, NANOG, MYC and POU5F1 were all down-regulated in human iPSC-derived atrial cardiomyocytes signifying that these cardiomyocytes are no longer pluripotent and are on the path to terminal differentiation (Figure 2A). This data confirms that key cardiac genes and contractile proteins (both shown in red) are up-regulated in the iPSC-derived atrial cardiomyocytes, relative to iPSC-derived ventricular cardiomyocytes (Figure 2B). Several atrial-specific genes (shown in dark blue) are also up-regulated in the iPSC-derived atrial cardiomyocytes compared to iPSC-derived ventricular cardiomyocytes from the same donor.
Importantly, the expression of atrial-specific ion channels KCNA5, KCNJ3 and KCNJ5 are also selectively increased in the iPSC-derived atrial cells compared to iPSC-derived ventricular cardiomyocytes, suggesting that they manifest the correct biophysics and pharmacology for atrial fibrillation drug discovery and disease modelling. In contrast, ventricular-specific genes (shown in yellow) are down-regulated in the iPSC-derived atrial cardiomyocytes, consistent with the creation of differentiated iPSC-derived atrial cardiomyocytes.
iPSC-derived atrial cardiomyocytes: biophysical and pharmacological characterisation
Ion channels and electrical signalling are fundamental aspects of healthy atrial cardiomyocyte function and atrial fibrillation disease processes. To confirm consistency, we also undertook biophysical and pharmacological validation of the iPSC-derived atrial cardiomyocytes using manual patch clamp electrophysiology recordings.
The biophysical features of the iPSC-derived atrial cardiomyocytes were determined using current clamp recordings of action potential (AP) parameters (Figure 3). At room temperature, the cells spontaneously beat and fired APs with a frequency of 0.3 Hz, facilitated by a negative resting potential of –73 mV and a robust upstroke velocity indicative of good Nav1.5 channel availability, yielding strongly overshooting AP amplitudes.
Figure 3: Biophysical characteristics of spontaneous iPSC-derived atrial cardiac APs. Data generated in collaboration with Metrion Biosciences.
The functional expression of relevant cardiac ion channels was confirmed using selective pharmacological ligands. The presence of the core panel of Nav1.5, Cav1.2 and hERG channels shown to be expressed in the iPSC-derived atrial cardiomyocytes by gene microarray studies (highlighted in Figure 2) was confirmed by the effects of the selective inhibitors lidocaine, nifedipine and E-4031 (Figure 4). These reagents produced the expected changes in AP upstroke and amplitude, highlighting the functionality of these ion channels. Furthermore, when applied to spontaneously beating iPSC-derived atrial cardiomyocytes, moderate concentrations of the hERG blocker dofetilide induced arrhythmia, an observation that is consistent with native human cardiomyocytes.
Figure 4: Core cardiac ion channel pharmacology of iPSC-derived atrial cardiomyocytes. Data generated in collaboration with Metrion Biosciences.
The atrial phenotype of these iPSC-derived cardiomyocytes was further confirmed by observing the characteristic effects of modulators of atrial-specific ionic currents shown to be up-regulated in the gene microarray data. For example, activation of the acetylcholine-activated inward-rectifying potassium current mimicked the negative chronotropic effect of vagal tone to slow spontaneous activity, while inhibition of the ultra-rapid delayed rectifier potassium current prolonged APD20. As such, the functional ion channel characteristics of these human iPSC-derived atrial cardiomyocytes strongly reflects that seen in native human tissue and confirms the expression of ion channel targets relevant to atrial fibrillation drug discovery.
Towards a more robust model of atrial fibrillation
Batch-to-batch consistency is essential in cell reagents and assay applications that will be used for drug discovery screening and disease modelling – the biophysical and pharmacological profiles of Axol’s human iPSC-derived atrial cardiomyocytes were found to be very consistent between pilot batches and commercial scale-up materials (for more information, see the new white paper). As such, they are likely to prove a very reliable source of cells for use in both.
Given the enormous potential of human iPSC-derived cells as translationally useful tools to enhance our understanding of atrial fibrillation, these impressive characterisation results demonstrate that Axol’s iPSC-derived atrial cardiomyocytes are a highly consistent and reproducible platform capable of underpinning a wide range of applications across drug discovery and preclinical research.
Download the whitepaper, developed in collaboration with Metrion Biosciences, to learn more about how our validated iPSC-derived atrial cardiomyocytes could help your research.