Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2), at concentrations that selectively block I(K1) (50-100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied, but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells, but robust expression of I(Kr.) In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.

Figure 1. Immuno-labelling of a beating cluster and single hiPSC-CM. Ai-Aiv

: Immuno-labelling of a beating cluster, exhibiting contractile activity prior to immunohistochemical processing, with Troponin T specific antibody to visualize cardiomyocytes and propidium iodide to visualize the nuclei of all cells in the BC. The scale bar represents 50 µm, B-E: Immuno-labelling of single cells dissociated from a BC with antibodies against canonical pan-cardiac specific marker- Troponin T with α-actinin (B), ventricular myocyte specific MLC-2v (C), atrial myocyte specific MLC-2a (D) and pacemaker specific HCN4 (E). Scale bars in B-E represent 20 µm.

Figure 2. Similarities among beating clusters derived from different embryoid bodies.

Similarities between spontaneous rate (BPM; beats/min) and APD₉₀ (including cAPD₉₀-B) obtained from stable action potentials recordings derived from 87 cardiac beating clusters (BC) (A and B; 17 batches of EBs) and from 27 BC derived from the same batch of EBs (C and D). A: Atrial-like; V: Ventricular-like. Atrial- and Ventricular-like action potentials were sorted out on the basis of the APD_30–40/APD_70–80 ratios (RO); RO >1.5 = Ventricular. cAPD₉₀-B: Bazett’s correction (ADP₉₀/(CL)^1/2). a: p<0.05; c: p<0.001 (vs. Atrial-like).

Figure 3. Representative Action Potentials derived from 27 beating clusters obtained from the same batch of embryoid bodies.

Action potential (AP) recordings obtained from 27 BC studied from the same batch of EBs. Individual AP parameters are tabulated in Table 2 . The traces are arranged by the number of days post-differentiation (Age). A: Atrial-like APs (15%); all others were classified as Ventricular-like (85%).

Figure 4. Electrophysiologic parameters as a function of age and cycle length (I). A to D

: Action potential parameters as a function of days post-differentiation (Age). E & F: APD₉₀ as a function of the cycle length (CL). CL range: 327 to 7063 msec; APD₅₀ range: 71 to 635 msec; APD₉₀ range: 70 to 789 msec; AP amplitude range: 58 to 121 mV; V_max range: 5 to 86 V/sec; (n = 103 BC). CL: Cycle Length; APD₅₀ and APD₉₀: Action potential duration at 50 and 90% repolarization, respectively; AP amplitude: Action potential amplitude; V_max: dV/dt of Phase 0.

Figure 5. Electrophysiologic parameters as a function of age and cycle length (II). A to D:

Action potential parameters as a function of dpd (Age). Relationship between maximum diastolic potential (MDP) or V_max and days pos-differentiation (Age) for 103 BC (A and B); 40 BC displaying atrial-like APs (C and D) and 63 BC displaying ventricular-like APs (E and F). The results indicate a significant increase in Vmax as a function of age (panels B and F) as well as a more negative in MDP, particularly in the early post-differentiation period (panels A and E; monoexponential fit).

Figure 6. Different electrophysiologic effects of E-4031 in two distinct populations of beating clusters.

E-4031-induced I_Kr block leads to EADs in cells from some beating clusters, but results in depolarization of cells in the majority of BC. A and B: Shown are action potential (AP), V_max and contraction (Edge Motion) recordings from a 69 day-old (A) and a 102 day-old (B) beating clusters under control conditions and following the addition of 5 µM E-4031. In A, E-4031 induced EADs within 5 min. In B, E-4031 led to depolarization within 3 to 4 min.

Figure 7. Different

electrophysiologic effects of BaCl₂ in two distinct populations of beating clusters. Shown are action potentials and V_max traces recorded from a 106 day-old (A) and a 105 day-old (B) beating cluster under control conditions and following the addition of 50, 100 and 500 µM BaCl₂. In A, 50 and 100 µM BaCl₂ led to membrane depolarization, consistent with the effect of BaCl₂ to block I_K1. In B, 50 and 100 µM BaCl₂ induced no changes in MDP, suggesting lack of I_K1. At a concentration of 500 µM, the APs of both beating clusters depolarized.

Figure 8. Concentration-dependence of the effect of BaCl₂ on AP amplitude and V_max.

Concentration-dependence of the effect of BaCl₂ to reduce AP amplitude, V_max and MDP in two population of beating clusters (BC). 100 µM BaCl₂ induced no changes in MDP in 13 out of 22 BC suggesting a small contribution or lack of I_K1 (A, C and E), but led to membrane depolarization in 9 out of 22 BC (B, D and F). At concentrations at which BaCl₂ also blocks I_Kr (500 µM), AP amplitude and V_max decreased in both groups of BC. a: p<0.05 vs. Control; c: p<0.001 vs. Control.

Figure 9. The different electrophysiologic effects of E-4031 and BaCl₂ is age-independent. A:

Range of days post-differentiation (Age) at which E-4031 (5 µM) induced EADs with little to no change in MDP vs. those at which it led to depolarization of BC without exhibiting EADs. B: Range of age of BC that depolarized in response to 100 µM BaCl₂ (Depolarization) vs. those that did not (No depolarization). Each point represents an individual BC; horizontal lines are the mean values for each group.

Figure 10. Characteristics of I_Kr in hiPSC-CM. A:

Representative current traces showing I_Kr recorded from hiPSC-derived cardiomyocytes in response to the voltage clamp protocol (top of figure). B: Mean I-V relation for I_Kr tail current (n = 11).

Figure 11. I_K1 is relatively low or absent in most hiPSC-CM. A

: Plot of inward rectifier potassium currents (I_K1) density in hiPSC-CM obtained at −100 mV hyperpolarizing pulse as a function of age post-differentiation. Red circles denote the mean for early (18 to 29; n = 16 cells), intermediate (35 to 74; n = 12 cells) and late (89 to 121; n = 10 cells) days post-differentiation (dpd), which are delimited by the blue. B: Effect of barium: Currents were recorded in hiPSC-CM of 121 dpd under control conditions (left) and in the presence of 500 µM barium (right). Currents were recorded during 400 msec pulses from −140 to 0 mV applied from a holding potential of −80 mV with a prepulse to −20 mV (The voltage protocol is shown in the inset). C: I-V relationship of barium-sensitive-I_K1 obtained by digital subtraction of currents recorded in the absence and presence of BaCl₂ in hiPSC-CM of 121 and 19–36 dpd. Data were normalized to cell size as reflected by capacitance measurements. Asterisks indicate statistically significant difference between groups (p<0.05).

Figure 12. I_Kr and I_K1 expression in hiPSC-CM and beating clusters. A-B:

I_Kr (KCNH2) and I_K1 (KCNJ2/Kir2.1) mRNA expression in hiPSC-derived BC at different stages of maturity. A: Relative expression levels of KCNJ2/Kir2.1, KCNJ12/Kir2.2, KCNJ4/Kir2.3 and KCNH2 mRNA from the pool of BC. The error bars represent standard error of the mean B:Percentage of Troponin T⁺ cardiomyocytes displaying protein expression of KCNH2 (hERG) and Kir2.1 C-D: Validation of hERG (I_Kr ) and Kir2.1 (I_K1) antibodies to determine their specificity in HEK293 cells transfected with respective cDNAs. E-F: Protein expression of hERG (I_Kr) and Kir2.1 (I_K1) in single enzymatically-dissociated cardiomyocytes from BC of 17 and 160 days post-differentiation as analyzed by immunohistochemistry. The majority of Troponin T⁺ hiPSC-CM showed little or no expression of KCNJ2 (I_K1), whereas over 90% of Troponin T⁺ cells showed hHERG/KCNH2 (I_Kr) expression at all stages of maturity. Representative Troponin T⁺ cells which stained either positive or negative for either I_Kr or I_K1 in the same immunoslide under identical imaging conditions are shown for cells dissociated from beating clusters 160 dpd. Scale bar represents 20 µm.

Figure 13. Mathematical model of hiPSC-CM APs.

Mathematical model demonstrating that significant reduction of I_K1 predicts a more depolarized MDP, the appearance of enhanced spontaneous phase 4 depolarization and automaticity as well as a critical dependence of MDP on I_Kr. A: Normal ventricular AP stimulated by the Luo-Rudy II model at a CL of 1000 msec. B: When I_K1 is decreased to 11% of the normal value, AP depolarizes and displays stable automatic activity (MDP is −53.6 mV; CL is 461 msec). C: Decreasing I_Kr to 50% of the normal value in the presence of 11% I_K1 results in further depolarization with EADs developing after 20 seconds. D: A larger block of I_Kr to 40% of the normal value elicits progressively decreasing oscillations of membrane potential leading eventually to the permanent depolarization at −12.8 mV.