Abstract
Dysfunction of the heart’s physiological pacemaker, the sinoatrial node (SAN), contributes to the onset of cardiac arrhythmias. Conventional treatment is accomplished by implantation of an electronic pacemaker, which involves a number of surgical risks and post-operative drawbacks for the patient. There is a need for the development of a clinically relevant biological pacemaker that overcomes ethical and immunogenicity concerns associated with embryonic stem cells as well as limitations with other biopacemaker strategies. This can be accomplished through utilizing human induced pluripotent stem cells (hiPSCs) as a platform to produce an autologous cellular therapy. Despite the generation of improved yields of cardiomyocytes (CMs) from the latest directed cardiogenesis protocols, the pacemaking cardiomyocyte (P-CM) population remains the smallest fraction of differentiated hiPSC-derived CMs (hiPSC-CMs). The activation of small conductance calcium-activated potassium (SK) channels in mouse embryonic stem cells has previously demonstrated to drive cardiac specification toward functional pacemaking-like cardiomyocytes. The native SAN is composed of P-CMs that arise from a population of precursor cells of the second heart field during embryonic development that characteristically express the Islet-1 (Isl1) transcription factor. The overall goal of this project was to derive an augmented P-CM population from hiPSCs through the pharmacological activation of SK channels on the day yielding the greatest percentage of Isl1+ cardiac progenitor cells (CPCs) during directed differentiation. Transcriptional qPCR analysis of cells between day 0-60 of cardiac differentiation revealed that SK2 is the dominant channel isoform expressed in hiPSC-CMs. The transcriptional expression of Isl1 was confirmed to be upregulated in differentiating cells between day 4-7 based on qRT-PCR data. Inhibition of canonical Wnt signaling was verified to be required during day 3-5 of differentiation to maintain this high level of Isl1 expression during this timeframe. Protein analyses through immunocytochemistry and flow cytometry identified day 8 to be the day of differentiation that yielded the highest percentage of Isl1+ CPCs. The small molecule modulation of SK channel activity in CPCs at day 8 of differentiation for 48 hours did not interfere with proper cardiac progression at the transcript level based on qPCR assessment. Electrophysiological characterization by confocal spinning disk microscopy of single hiPSC-CMs subjected to SK channel activation showed preliminary evidence of increased commitment toward nodal and pacemaking CMs. These findings suggest that the pharmacological activation of SK channels in Isl1+ CPCs may represent a promising approach for the optimal induction of P-CM subtype specification during directed cardiac differentiation.