Introduction: The sinoatrial node (SAN) is the intrinsic pacemaker of the heart. It harbors pacemaker cells that spontaneously depolarize and initiate electrical signals through the heart’s conduction system, triggering the heart to beat. Impaired SAN function disrupts the natural pacing of the heart and results in abnormal heart rhythms called arrhythmias. The current approach to treating cardiac arrhythmias involves using a surgically-implanted electronic pacemaker that works to compensate for irregular heart rhythms and initiate pacing when necessary. Despite its effectiveness, device pacemaking is costly, susceptible to infection, and limited in battery life. In search of a minimally invasive way to treat arrhythmias, biological pacing has emerged as a potential solution. In fact, biological pacemakers derived from stem cells could serve as an alternative therapy for cardiac arrhythmias. Stem cells can be differentiated into ventricular and atrial myocytes (VMs and AMs), but it remains unclear whether differentiated AMs can exhibit pacemaker-like action potentials. Consequently, studying the beating and propagation behavior of these myocytes is essential for developing stem cell-derived biological pacemakers. This study aims to utilize optical mapping to determine if human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes can produce ventricular-like and atrial-like action potentials, to investigate whether differentiated AMs exhibit pacemaker-like action potentials, and to observe the behavior of these myocytes when treated with the pacemaker channel (HCN) blocker Ivabradine. It was hypothesized that optical mapping can help detect action potential differences between the differentiated VMs and AMs, and AMs will display a chronotropic response to Ivabradine.
Materials and
Methods: WTC11 hiPSCs were differentiated to cardiac lineage by treatment with the WNT signaling pathway activator CHIR99021 on day 0 (d0) to induce mesodermal specification, followed by treatment with the WNT signaling inhibitor IWR1 on d2 to promote the cardiomyocyte phenotype. For atrial differentiation, retinoic acid (RA) was added on d3 to form a heterogeneous population of pacemaker and atrial myocytes, with fresh RA replaced on d5 and washed out on d6. Atrial cells were maintained until d24 for optical mapping. For ventricular differentiation, no additional small molecules were added, and cells were maintained until d28 for optical mapping. Single cell flow cytometry was performed on d17 of differentiated VMs and AMs to assess cardiomyocyte efficiency by detecting double positive expression of cardiac troponin T (cTnT) and alpha-actinin 2 (ACTN2).
For optical mapping, d28 VMs and d24 AMs were loaded with the voltage-sensitive dye Fluovolt and myosin II inhibitor Blebbistatin to reduce motion artifacts, which were washed out after 20 minutes. Cells were loaded with Gey’s Balanced Salt Solution (GBSS) containing Blebbistatin for imaging. Ivabradine treatment involved adding 1uM Ivabradine to the GBSS solution. The experimental setup consisted of the MiCAM03-N256 Single Camera System vertically connected to a longpass emission filter (580LP), 50mm objective lens, and a 495LP dichroic filter cube. Samples were excited with a blue light source (470nm), and signals were captured at 1,000 frames per second and spatial resolution of 256x256 pixels. Brainvision (BV) Workbench software was used to analyze raw optical mapping recordings.
Results, Conclusions, and Discussions: Flow cytometry analysis of WTC11-derived VMs and AMs revealed that VMs are 88% double positive for cTnT and ACTN2, and AMs are 46% double positive on average (Figure 1), indicating robust cardiomyocyte efficiency for both VMs and AMs. Optical mapping showed that VMs had smooth electrical propagation with depolarization originating from the left side of the well and spreading rightward. However, AMs displayed distinct activation of localized activity with the activation wavefront splitting into two directions from one source, leading to a non-uniform activation pattern (Figure 2A). In addition, VMs unexpectedly lacked the characteristic plateau phase and exhibited a slower rise time compared to AMs, which had typical atrial-like triangular action potentials with a faster rise time (Figure 2A). Additionally, AMs had a higher spontaneous beating rate than VMs (Figure 2B). Characterization of the WTC11-derived VMs and AMs revealed that AMs highly correspond to the human atrial subtype but VMs did not completely display similar properties of human ventricular cardiomyocytes. Ivabradine treatment reduced the beating rate in both AMs and VMs (Figure 2B). In VMs, the activation map retained a unidirectional propagation pattern but showed longer activation times. AMs, however, had a different activation pattern, with the earliest activation originating from a different region of the well and less widespread activity (Figure 2C).
Overall, WTC11-derived VMs and AMs demonstrated distinct action potential profiles and responses to Ivabradine. VMs showed unchanged propagation behavior but a decreased beating rate, but also a round-like action potential morphology when recorded spontaneously and treated with Ivabradine. AMs exhibited the expected triangular-like action potentials and reduced beating rate with Ivabradine, though pacemaker-like action potentials were not observed, possibly due to a smaller pacemaker population within the AMs. The reduction in the beating rate of VMs may be attributed to the presence of the AM population within them. Optical mapping distinctly identifies the electrophysiological characteristics of WTC11-derived VMs and AMs, however, further studies are needed. Specifically, qPCR for gene expression analysis of ventricular, atrial, and pacemaker genes will provide a deeper understanding of their phenotypes and the electrophysiological behavior observed in optical mapping.
