Human iPSC-Derived Cardiomyocytes

Flow Cytometry and Immunofluorescence Analysis of hiPSC-CMs

Figure 1A shows representative flow cytometry data to assess cardiomyocyte purity. "Live" (live/dead stain) permeabilized cardiomyocytes were stained for cTNT or matching isotype control. For each analysis, the cardiomyocyte cultures contained cTNT-positive cells with a purity of >98%, and after 2 week in culture, cardiomyocyte purity remained unchanged. Figure 1B shows a cardiomyocyte monolayer immunostained for sarcomeric α-actinin (red) and nuclei (blue). Nuclei are evenly distributed and α-actinin staining is present throughout the field. The higher power image (inset) shows the subcellular sarcomeric organization of contractile myofibrils.

Fig. 1. Purity and structure of cardiomyocytes (Ma, Junyi, et al., 2011).

Action Potentials (APs) Properties of hiPSC-CMs

APs from 59 cardiomyocytes that beat spontaneously at a constant rate were analyzed for beat rate interval (Interval), maximum diastolic potential (MDP), peak voltage (Peak), amplitude (Amp), maximal rate of depolarization (dV/dt_max), and AP duration (APD) at different levels of repolarization (APD measured at 10% increments of Amp). These data are summarized in Fig. 2. The following criteria were used to distinguish the AP phenotypes: atrial- and ventricular-like APs displayed more negative MDPs with higher dV/dt_max values and larger amplitudes than nodal-like APs. Ventricular-like APs have a distinct plateau phase (phase 2) after which repolarization accelerates (phase 3). This was measured as the time difference between APD₃₀ to APD₄₀ (APD_30-40) and the time difference between APD₇₀ to APD₈₀ (APD_70-80). Ventricular-like APs had a ratio (APD_30-40/APD_70-80) of ∼2.5 compared with the atrial-like ratio of ∼1.1, thus, APs with a ratio >1.5 were categorized as ventricular like. Ventricular-like APs had longer duration APDs with slower rates of spontaneous beating (increased Interval), whereas atrial-like APs had a minimal plateau phase (APD_30-40/APD_70-80 close to unity) with shorter durations and higher spontaneous beating rates. Nodal-like APs had less negative MDPs and smaller amplitudes with lower dV/dt_max values (<10 V/s). With the use of these criteria, 54% of the 59 cardiomyocytes displayed ventricular-like APs, 22% showed nodal-like APs, and 24% showed atrial-like APs.

Fig. 2. Action potentials (APs) were recorded from hiPSC-CMs at 35-37°C using the perforated patch-clamp method (Ma, Junyi, et al., 2011).

The effects of selective ion channel block were tested on cardiomyocytes with ventricular-like AP waveforms stimulated at a constant rate of 1 Hz. For control experiments, AP parameters were stable over a 15-min recording period in extracellular solution without (Fig. 3A, left) or containing 0.1% DMSO (Fig. 3B, left). Selective block of I_Na by TTX had no effect on AP parameters at 1 μmol/L, whereas increasing concentrations (3-30 μmol/L) delayed the upstroke and reduced dV/dt_max (Fig. 3A, middle). Selective block of the I_Kr by E4031 caused concentration-dependent (3-100 nmol/L) slowing of repolarization to "triangulate" the AP (Fig. 3A, right). Selective block of I_Ca by nifedipine caused concentration-dependent (3-100 nmol/L) shortening of the AP throughout repolarization with minimal effects on the AP peak voltage and dV/dt_max (Fig. 3B, middle). Suppression of the I_Ks by 3R4S-chromanol 293B had minimal effects on AP properties up to 10 μmol/L sufficient to cause >50% block of I_Ks (Fig. 3B, right). For measurements of APD, the time of dV/dt_max was used as the start time for the AP. TTX caused slowing of dV/dt_max, E4031 caused lengthening of APD₅₀ and APD₉₀, and nifedipine caused shortening of APD₁₀, APD₅₀, and APD₉₀. Single EADs were induced by 30-100 nmol/L E4031 in three of five cardiomyocytes paced at 0.5 Hz. Figure 3C shows that lower concentrations of E4031 prolonged the AP and that 100 nmol/L induced single EADs. Similar to cardiac myocytes, EADs in hiPSC-derived cardiomyocytes were rate dependent and disappeared upon increasing the pacing rate from 0.5 to 1 Hz. Furthermore, there was an inverse relation between the EAD peak voltage and EAD take-off potential, as is shown in Fig. 3D for 79 single EADs from the same cardiomyocyte (inset shows 2 EADs with differing amplitudes and take-off potentials).

Fig. 3. Effects of ion channel blocking drugs on AP morphology. Data were recorded at 35–37°C with the perforated patch-clamp method (Ma, Junyi, et al., 2011).