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

<p>Range of days post-differentiation (Age) at which E-4031 (5 µM) induced EADs <i>with little to no change in MDP</i> vs. those at which it led to depolarization of BC <i>without exhibiting EADs</i>. <b>B:</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.</p

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

<p>: 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>B-E:</b> 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 (<b>C</b>), atrial myocyte specific MLC-2a (D) and pacemaker specific HCN4 (<b>E</b>). Scale bars in <b>B-E</b> represent 20 µm.</p

Summary data of the effect of E-4031.

<p>Electrophysiologic parameters measured under control conditions and following 5 µM E-4031 from beating clusters in which this intervention led to EADs (A, n = 8 [29 to 116 days old]) and from those in which it did not (B; n = 13 [25 to 118 days old]). Values are means ± SD. a: p<0.05 vs. Control; b: p<0.001 vs. B; c: p<0.01 vs. Control; d: p<0.005 vs. Control; e: p<0.05 vs. B; f: p<0.001 vs. Control; g: p<0.01 vs. B.</p

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

<p>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 (<b>A, </b><b>C and E</b>), but led to membrane depolarization in 9 out of 22 BC (<b>B, D and F</b>). 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.</p

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

<p>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. <b>A and B</b>: Shown are action potential (AP), V_max and contraction (Edge Motion) recordings from a 69 day-old <b>(A)</b> and a 102 day-old <b>(B)</b> beating clusters under control conditions and following the addition of 5 µM E-4031. In <b>A</b>, E-4031 induced EADs within 5 min. In <b>B</b>, E-4031 led to depolarization within 3 to 4 min.</p

Mathematical model of hiPSC-CM APs.

<p>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. <b>A:</b> Normal ventricular AP stimulated by the Luo-Rudy II model at a CL of 1000 msec. <b>B:</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). <b>C:</b> 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. <b>D:</b> 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.</p

Electrophysiologic parameters from 27 BC of the same batch of EBs.

<p>Control action potential parameters derived from stable recordings obtained from 27 BC of the same batch of EBs. The information is sorted by the APD_30–40/APD_70–80 ratio (from the smallest [top] to the largest [bottom]). Age: number of days post-differentiation. The corresponding AP traces are pictured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040288#pone-0040288-g002" target="_blank"><b>Figure 2</b></a>. cAPD₉₀-B (Bazett’s correction); cAPD₉₀-H (Hodge’s correction); cAPD₉₀-Fri (Fridericia’s correction); cAPD₉₀-Fra (Framingham correction).</p

RT-PCR primer sequences used in this study.

<p>RT-PCR primer sequences used in this study.</p

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

<p> 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).</p

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

<p>I_Kr (KCNH2) and I_K1 (KCNJ2/Kir2.1) mRNA expression in hiPSC-derived BC at different stages of maturity. <b>A:</b> 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>B:</b>Percentage of Troponin T⁺ cardiomyocytes displaying protein expression of KCNH2 (hERG) and Kir2.1 <b>C-D:</b> Validation of hERG <b>(</b>I_Kr<b>)</b> and Kir2.1 (I_K1) antibodies to determine their specificity in HEK293 cells transfected with respective cDNAs. <b>E-F:</b> 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.</p