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

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (I_K1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial I_K1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic I_K1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon I_K1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic I_K1. In conclusion, the dynamic clamp-based approach of I_K1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

Keywords: cardiomyocytes; computer simulations; differentiation; electro-physiology; induced pluripotent stem cells; inward rectifier potassium current; perforated patch-clamp; retinoic acid.

Figure 1

Superimposed action potentials of control (CTRL) and retinoic acid-treated (RA-treated) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). (A,B) Typical action potentials of (A) fast beating and (B) slowly beating CTRL and retinoic acid (RA)-treated hiPSC-CMs. (C) Typical action potentials during overdrive stimulation at 1 Hz. Note the expanded time scale.

Figure 2

Experimental approach to supply a patched hiPSC-CM with a synthetic inward rectifier potassium current (I_K1). (A) Diagram of the experimental setup. The Real-Time Linux (RT-Linux)-based PC computes the synthetic I_K1 according to the membrane potential V_m that is read into the PC. This I_K1 is sent to the patch-clamp amplifier, operating in current clamp mode, which adds any stimulus current and injects the net current (I_inj) into the patched hiPSC-CM. This process is updated with a time step ∆t. (B) Current–voltage relationship of the synthetic I_K1. E_K is the potassium equilibrium potential.

Figure 3

Effect of I_K1 injection on the action potential of hiPSC-CMs. (A,B) Action potential of (A) a CTRL and (B) an RA-treated hiPSC-CM in the absence and presence of a synthetic I_K1, which is computed in real time according to the current–voltage relationship of Figure 2B and supplied through the patch-camp pipette. Insets: time derivative of the AP upstroke. (C,D) Corresponding dynamic clamp current injected into the cell. The sharp cut-off peak of 3 ms duration starting at 5 ms represents the stimulus current, which is applied at 1 Hz.

Figure 4

Standard box plot of the AP plateau amplitude of all CTRL and RA-treated hiPSC-CMs measured (n = 13 and n = 18, respectively), both with and without I_K1 injection.

Figure 5

Standard box plots of action potential parameters of all CTRL and RA-treated hiPSC-CMs measured (n = 13 and n = 18, respectively), both with and without I_K1 injection. (A) Maximum diastolic potential (MDP). (B) Maximum upstroke velocity (V_max). (C) Action potential amplitude (APA). (D) Action potential duration at 20% repolarisation (APD₂₀). (E) Action potential duration at 50% repolarisation (APD₅₀). (F) Action potential duration at 90% repolarisation (APD₉₀).

Figure 6

Variability in action potential parameters of the CTRL and RA-treated hiPSC-CMs, both with and without I_K1 injection. (A) Standard deviation (SD) and (B) coefficient of variation (CV) of the AP parameters of the CTRL hiPSC-CMs. (C) SD and (D) CV of the AP parameters of the RA-treated hiPSC-CMs. MDP: maximum diastolic potential; V_max: maximum upstroke velocity; APA: action potential amplitude; Plateau: AP plateau amplitude; APD₂₀, APD₅₀, and APD₉₀: action potential duration at 20%, 50%, and 90% repolarisation, respectively. * p < 0.05.

Figure 7

Electrical activity of the ventricular-like and atrial-like hiPSC-CM model cells of Paci et al. [30,31]. (A) Spontaneous action potentials. (B) Action potentials and (C) associated intrinsic inward rectifier potassium current (I_K1) during 1 Hz overdrive stimulation on an expanded time scale.

Figure 8

Current added to the ventricular-like and atrial-like hiPSC-CM model cells of Paci et al. [30,31] in order to obtain a resting membrane potential near −80 mV. 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].

Figure 9

Electrical activity of simulated ventricular-like and atrial-like hiPSC-CMs. (A,B) Action potential during 1 Hz stimulation of (A) a ventricular-like and (B) an atrial-like model cell in the absence and presence of an additional I_K1-like current, as applied by Meijer van Putten et al. [5], Bett et al. [14], or Rocchetti et al. [15], or a hyperpolarising current of constant amplitude, as applied by Jara-Avaca et al. [16]. Insets: maximum AP upstroke velocity. (C,D) Associated additional current.