Abstract Body: Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit spontaneous automaticity, which contributes to post-transplantation arrhythmias. While "membrane" (HCN channels) and "calcium" (SR-calcium release/NCX) clocks drive this activity, their pharmacological inhibition often fails to completely abolish spontaneous beating, suggesting additional regulatory mechanisms.
Hypothesis: We hypothesized that the mechanosensitive ion channel Piezo1 is expressed in hiPSC-CMs and modulates spontaneous automaticity and calcium handling independent of canonical pacemaker currents.
Goals: To detect Piezo1 expression in hiPSC-CMs and characterize its functional contribution to spontaneous automaticity and calcium spark frequency.
Methods: Piezo1 expression was evaluated in hiPSC-CMs (D20-26) via Western Blot (WB). Spontaneous frequency and calcium transients (Fluo-4) were measured after Piezo1 selective agonist Yoda1 stimulation (0.1-20 µM). Pathway independence was assessed during HCN blockade (Ivabradine, 10 µM) or ß-adrenergic blockade (Propranolol, 10 µM). Spontaneous SR-calcium release (sparks) was quantified using high-speed confocal microscopy. Data are Mean ± SEM; analyzed by ANOVA or t-test (P < 0.05 significant).
Results/Data: WB confirmed Piezo1 expression in hiPSC-CMs. Compared to Vehicle, Yoda1 induced a significant, dose-dependent increase in frequency (1.13 ± 0.02 vs. 2.01 ± 0.11; P < 0.0001). This persisted independently during both HCN and ß-adrenergic inhibition, suggesting an independent excitatory pathway. Piezo1 activation increased calcium release velocity (1.00 ± 0.08 vs. 1.35 ± 0.12; P = 0.03) and shortened contraction time (0.59 ± 0.03s vs. 0.44 ± 0.02s; P = 0.0002). Furthermore, Yoda1 (2.5 µM) significantly elevated spark Frequency (4.08 ± 0.35 vs. 11.68 ± 0.83 sparks/100 µm/s; P < 0.0001) and altered spark width (FWHM: 0.99 ± 0.05 µm vs. 1.65 ± 0.09 µm; P = 0.0002).
Conclusion(s): These findings identify Piezo1 as a functional component of the hiPSC-CM "coupled-clock." The persistence of its effects under canonical blockade suggests that mechanotransduction directly modulates SR calcium leak, potentially serving as a trigger for spontaneous activity and a target for mitigating post-transplantation engraftment arrhythmias.