EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival

Abstract

SummarySubtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1–4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

“…Bett et al [ 14 ] and Rocchetti et al [ 15 ] also employed an “electronic expression” of I K1 in their patch-clamp experiments on hiPSC-CMs, but they used different I K1 characteristics, as described in detail in Section 4.5 . Others, like Jara-Avaca et al [ 16 ], injected a hyperpolarising current of constant amplitude to obtain an RMP near −80 mV. We were interested to what extent the characteristics of the injected current would affect the outcome of the experiments.…”

“…We tested this, but not in patch-clamp experiments on hiPSC-CMs, because we anticipated that the use of real hiPSC-CMs, with their considerable variability in AP morphology ( Figure 4 , Figure 5 and Figure 6 ), might lead to ambiguous results. Instead, we ran computer simulations with models of ventricular-like and atrial-like hiPSC-CMs [ 30 , 31 ], to which we added an outward current with the characteristics of the one used by Meijer van Putten et al [ 5 ], Bett et al [ 14 ], Rocchetti et al [ 15 ], and Jara-Avaca et al [ 16 ].…”

“…Figure 8 shows the I K1 current–voltage relationships of Meijer van Putten et al [ 5 ], Bett et al [ 14 ], and Rocchetti et al [ 15 ] as well as the hyperpolarising current of Jara-Avaca et al [ 16 ]. The current–voltage relationships employed by Meijer van Putten et al [ 5 ] and Rocchetti et al [ 15 ] appear to be highly similar.…”

“…In short, a synthetic I K1 is computed in real time based on the acquired membrane potential and injected into the hiPSC-CM through the patch-clamp pipette, thus hyperpolarising the cell and stabilizing its RMP and improving the resemblance with the AP configuration of atrial or ventricular cardiomyocytes. Another “electronic” solution to obtain an RMP near −80 mV, not requiring a dynamic clamp system, is to inject a hyperpolarising current of constant amplitude, as applied by, e.g., Jara-Avaca et al [ 16 ].…”

Human pluripotent stem cell models of cardiac disease: from mechanisms to therapies

“…Similarly, modulating the retinoic-acid and Wnt signalling pathways during early stages of differentiation led to hESC- and hiPSC-CMs being directed to either an atrial-like or ventricular-like subtype ( Devalla et al, 2015 ; Karakikes et al, 2014 ; Zhang et al, 2011 ). Finally, it was recently reported that timed supplementation of the chemical compound 1-ethyl-2-benzimidazolinone (EBIO) increased the number of cardiomyocytes with nodal- and atrial-like phenotypes ( Jara-Avaca et al, 2017 ).…”