Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Aim Hydrogen sulfide (H2S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H2S. Methods and Results Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H2S, on action potential (AP), outward rectifier potassium currents (I Ks and I Kr), L-type Ca2+ currents (I CaL) and hyperpolarization-activated inward current (I f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I K (I Ks and I Kr), I CaL and I f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties. Conclusions This is the first demonstration of effects of H2S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H2S on I CaL and revealed additional novel inhibitory effects on I f, I Ks and I Kr currents in human cardiomyocytes.


Introduction
Safety and efficacy evaluation of pharmaceuticals for cardiac indications are hindered due to a shortage of suitable human in vitro cellular models. Such limitation has in part precipitated in market withdrawal of several cardiac as well as non-cardiac drugs recently [1,2]. Current available cardiotoxicity screening platform that relying on animal-derived cardiomyocytes or cell lines ectopically expressing ion channels are not ideal due to species difference in the former and inadequate ion channel interactions in the latter [3]. Human pluripotent stem cell (hPSC) including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSCs) are capable of cardiomyogenesis [4,5]. hESC-and hiPSC-derived cardiomyocytes (hESC-CMs and hiPSC-CMs) present an unprecedented window for an expedited evaluation of chemical entities for known cardiac toxicity or hitherto unknown cardiac implications. In contrast to hESCs, hiPSCs are capable of giving rise to a renewable source of cardiomyocytes from individual patients.
These patient-derived cardiomyocytes offer an immensely valuable resource in evaluating individual-specific responses towards pharmaceutical agents especially anti-arrhythmics that have a narrow therapeutic index that are often compounded by idiosyncratic patient response.
Hydrogen sulfide (H 2 S) is a recently identified physiological gaseous molecule associated with cardiovascular benefits such as vasodilation and cardiac protection [6]. It is known to affect multiple ion channels that could have implication in cardiac arrhythmia [7]. However, its influence on electrical remodeling of human cardiomyocytes is yet to be understood and its effect on individual ventricular, atrial and nodal cardiomyocyte subtypes has not been explored.
In this study, we demonstrated that H 2 S altered the action potential (AP) properties of hESC-and hiPSC-derived ventricular (V)-, atrial (A)-like CMs. H 2 S not only blocked L-type Ca 2+ current (I CaL ), but also showed inhibitory effects on the slow and rapid outward rectifier K + currents (I Ks and I Kr ).

Characterization of Human Pluripotent Stem Cell-derived Cardiomyocytes
Human iPSC-derived cardiomyocytes (hiPSC-CMs) were acquired from Cell Dynamic International (CDI, Wisconsin, USA) and they have been well characterized [10]. All cardiomyocytes used in this study were 5 weeks post cardiac differentiation. For structural characterization, cardiomyocytes were seeded on glass coverslips and immunocytochemically stained for sarcomeric cardiac markers using antibodies against cardiac sarcomeric aactinin (clone EA-35. Sigma), b-myosin heavy chain or b-MHC (Alexis Biochemicals, FL, USA) followed by Alexa FluoH 488 goat anti-rabbit IgG (Invitrogen, CA, USA); and cardiac Titin (1:10) (Sigma-Alrich, MO, USA) followed by Alexa FluoH 555 donkey anti-rabbit IgG. For electrophysiological characterization, H9 hESC-CMs and hiPSC-CMs were identified with spontaneous action potential (AP) recorded by Patch-Clamp technology later described. Ventricular (V)-, atrial (A) -and nodal (N)-like subtypes of cardiomyocytes were determined by their characteristic AP properties [11]. Evaluation of the Effect of H 2 S on hESC-and hiPSC-CMs Freshly prepared NaHS stock solution (1000 mM) was added to cardiomyocytes using a micro-perfusion system. For the concentration curve, NaHS was sequentially added to the cells from low to high concentrations ranging from 100 to 1500 mM.

Patch-clamp Electrophysiology
Cardiomyocytes seeded on 3.5 cm diameter petri dishes were transferred to a recording chamber mounted on the stage of an inverted microscope (TE2000-S, Nikon, Tokyo, Japan). Whole-cell AP and ion currents were recorded with a Patch-Clamp amplifier (Axon 200B, Axon Instruments, Foster City, CA, USA). Patch pipettes were fabricated with a Sutter P-97 horizontal puller (Sutter Instrument, Novato, CA) and had a resistance of 2-4 MV when filled with the internal solution. In experiments, 70-90% series resistance was compensated. Currents and voltage protocol generation, data acquisition and analysis, were performed using Clampex and Clampfit software (version 10.0, Axon Instruments). Except Ca 2+ current measurement, all experiments were performed at 37uC.
Measurement of outward K + current. In the voltageclamp mode, the outward K + current was measured on the same V-like cardiomyocytes after AP recording. Immediately after the AP measurement, Ca 2+ and Na + currents (I Ca and I Na ) were blocked with 0.5 mM CdCl 2 and 20 mmol/L TTX in normal Tyrode's solution, respectively. The outward K + currents were elicited by a 500-ms depolarization from holding potential of 280-mV to voltages ranging from 240 to +70 mV in 10-mV steps. Pipette solution for AP and I K (in mM): KCl 130, MgCl 2 1, MgATP 3, EGTA 10, and HEPES 10, pH 7.2 with KOH. Modified extracellular solution for the AP and outward K + currents (in mM): normal Tyrode's solution plus 0.5 mM CdCl 2 and 20 mmol/L TTX to block I Ca and I Na , respectively.
Measurement of L-type Ca 2+ currents. The H9 hESC-CM clusters contain homogeneous V-like cardiomyocytes (after consistent identification of 5 V-like cardiomyocytes in that cluster) were subsequently used for measuring of L-type Ca 2+ currents (I CaL ). The I CaL was evoked by a 400-ms pulse to +60 mV from the holding potential of 240 mV in 10 mV increment. Pipette solution for I CaL was prepared (in mM): CsCl 120, MgCl 2   The pipette solution contained (in mM) KCl 150, K 2 ATP 1, MgCl 2 5, and HEPES 3 (pH 7.2 with KOH). Cells were hyperpolarized from a holding potential of 240 mV to test potentials of 250 mV to 2110 mV for 3000 ms to elicit currents, which were completely blocked by CsCl (2 mM).

Measurement of Confocal Ca 2+ Transients
Calcium imaging using confocal fluorescent microscope was conducted in combination with AP recordings. Contracting clusters of homogenous H9 hESC-derived V-CMs on 3.5-cm glass bottomed dishes were identified and labeled after a consistent recording of over 5 CMs showing homogenous AP patterns of V-CM or N-CM. After 2 hours recovery in culture medium, CMs were loaded with 6 mg/mL Fluo-4 AM (Molecular Probes) for 15 min at 37uC and changed to normal Tyrode solution. Ca 2+ transients were recorded by a LSM-710 laser scanning confocal microscope (Carl Zeiss, Inc Germany) with a 6 40 oil immersion objective, numeric aperture = 1.3. Fluo-4 was excited at 488 nm using a 25 mW argon laser (with intensity attenuated to 1%). Fluorescence emission was measured at .505 nm. Images were acquired in the line (X-T)-scan mode with 512 pixels per line at a rate of 3 ms per scan. The scan line was oriented along the longitudinal axis of the cell, at pixel intervals of 0.15 mm. The axial resolution was set at 1.5 mm according to the manufacture's specifications. In some experiments, Ca 2+ transients were measured with the confocal microscope operating in the frame (X-Y) imaging mode. Ca 2+ images were analyzed using a computer program written in IDL 5.4 software [12].

Statistical Analysis
For ion current density, data were presented as mean6SEM (standard error of the mean) and reflected measurements of multiple cells. For the rest, data were expressed as mean6SD. Data were analyzed by 2-tailed Student t tests. P values below 0.05 were considered to indicate a statistically significant difference.

Cardiac Differentiation of H9 hESCs and Subsequent Characterization
Contracting EBs were obtained from H9 hESCs after 15 days of cardiac differentiation with differentiation efficiency (contracting EBs vs. total EBs) ranging from 2963% to 5065%, which is consistent with previous reports [13]. Dissociated hESC-CMs showed sarcomeric protein expression similar to those in previous reports (Fig. 1A). [4,5] Among dissociated hESC-CMs, ventricular (V)-, atrial (A)-and nodal (N)-like subtypes (Fig. 1B1) were identified by corresponding action potential (AP) properties which included action potential amplitude (APA), 50% and 90% of action potential duration (APD50 and APD90), and maximal rate of depolarization or maximal upstroke velocity (dV/dt max ) (Fig. 1B2). However, unlike adult quiescent CMs, A-and V-like hESC-and hiPSC-CMs were capable of spontaneous contractions in culture. The proportion of 3 subtypes of CMs from H9 hESCs was similar to previous reports with V-like CMs constituting the majority subtype (60,70%) of total cardiomyocyte populations [10,11].

Effect of H 2 S on hPSC-derived Cardiomyocytes
NaHS (a H 2 S donor) exerted a dose-dependent (from 100 to 1500 mM) effect on both H9 hESC-and hiPSC-CMs. Fig. 2 shows that NaHS dose-dependently prolonged the action potential duration of a H9 hESC-derived atrial-like CMs ( Fig. 2A) and both APD50 and ADP90 were significantly increased with approxi-mately 50% APD prolongation (IC 50 ) achieved at 300 mM (Fig. 2B). As the H 2 S solution contains a concentration that is approximately 33% of the original concentration of NaHS [14], 300 mM of NaHS is equivalent of ,100 mmol/L of H 2 S. Counting on the rapid decay of H 2 S under in vitro condition, this concentration is close to the top limit of the physiological concentration of H 2 S which is 50,90 mM [15,16]. Accordingly, the subsequent experiments were performed mostly with 100 mM of NaHS and 300 mM of NaHS was used to test the maximal effects. Figure 4. I K response of hESC-and hiPSC-CMs towards H 2 S. A, B: I K recorded on V-CMs derived from H9 hESCs (A) and hiPSCs (B) and A1, B1: I K recorded before and after NaHS (300 mM) treatment. A2, B2: I K recorded at baseline, with pre-treatment with a mixture of 5 mM E-4031 and 5 mM Chromanol 293B, and with subsequently treatment with NaHS (100 mM). A3, B3: I K recorded at baseline, with pre-treatment with 5 mM Chromanol 293B, and with subsequently treatment with NaHS (100 mM). A4, B4: I K recorded at baseline, with pre-treatment with 5 mM E-4031, and with subsequently treatment with NaHS (100 mM). A5, B5: Quantification of I K response of hESC-CMs (A5) and hiPSC-CMs (B5) towards Chromanol 293B, E-4031 and NaHS observed in A1,A4 and B1,B4, respectively. Relative quantity (% of baseline I K ) of Chromanol 293B-, E-4031-and NaHS-sensitive I K were calculated by subtracting post-treatment I K from baseline. *P,0.05; **p,0.01 (NaHS or I K blocker(s) treated vs. baseline). p,0.05 (NaHS treated vs. I K blocker treated). C/E: Chromanol 293B+E-4031; C: Chromanol 293B; E: E-4031. Number of repeats (n) = 6. doi:10.1371/journal.pone.0050641.g004

Action Potential Response of hPSC-CMs towards NaHS
Exposure to NaHS evoked distinct electrical responses from the 3 different subtypes of cardiomyocytes indicated by altered action potential properties. Following NaHS exposure, beating rates were significantly reduced in V-and A-like CMs derived from H9 hESC (p,0.05) and hiPSC (p,0.05) (Fig. 3A). No difference was found with N-like CMs (data not shown).
APDs in the V-and A-like CMs derived from H9 hESCs and hiPSCs were significantly prolonged following NaHS exposure. There was a significant prolongation of APD in the V-like CMs derived from H9 hESCs (APD50:504.7617. 8 . 3C). However, no statistically significant changes in APD were observed in the N-like CMs derived from H9 hESCs and hiPSCs (data not shown). In addition, no significant changes in action potential amplitude (APA), maximal upstroke velocity (dV/ dt max ) and maximum diastolic potential (MDP) were found following NaHS treatment (data not shown).

Ion Currents Response of hESC-and hiPSC-derived CM towards H 2 S
To investigate the mechanism behind the effect of H 2 S on prolongation of APD and slowing down of the contraction rates, effects of H 2 S in altering ion currents in H9 hESC-and hiPSCderived V-like CMs were investigated.

Exposure to NaHS Significantly Reduced Outward Potassium Currents (I K ) in H9 hESC-and hiPSC-derived CMs
Outward rectifier potassium currents (I K ) including slow and rapid I K (I Ks and I Kr ) contribute to the 2 nd and 3 rd phase of AP. With a modified protocol that facilitates the combined measure- ment of potassium currents with action potential, I K was recorded in V-and A-like CMs identified by AP measurement. Fig. 4 showed representative data recorded with V-like CMs.
It was found that exposure to NaHS (100 to 300 mM) significantly reduced total I K in both hESC-CMs (Fig. 4A) and hiPSC-CMs (Fig. 4B). The current-voltage (I2V) relationships demonstrated a suppression of NaHS (300 mM) on I K density (pA/ pF) (Fig. 4A1 and Fig. 4B1). However, such effect of NaHS on I K was found to be abolished after pretreatment of cardiomyocytes with a mixture of I K blockers containing 5 mM E-4031 (blocks I Kr ) and Chromanol 293B (blocks I Ks ) ( Fig. 4A2 and Fig. 4B2). Separately, the effects of NaHS on I Ks and I Kr were independently validated. Fig. 4A3 and Fig. 4B3 show that Chromanol 293B (a I Ks blocker) alone suppressed I K in both hESC-derived and iPSCderived V-like CMs respctively. Addition of NaHS to Chromanol 293B treated cells led to further attenuation of I K to a level close to that of NaHS treatment alone, suggesting that NaHS could additionally inhibit I Kr . Similarly, Fig. 4A4 and Fig. 4B4 show that E-4031 (a I Kr blocker) suppressed I K in both hESC-derived and hiPSC-derived V-like CMs respectively. Additional NaHS treatment resulted in a further reduction in I K to a level close to NaHS treatment alone, suggesting that NaHS could additionally inhibit I Ks . The effects of NaHS on I Ks and I Kr in hESC-CMs and hiPSC-CMs were quantified and summarized in Fig. 4A5 and Fig. 4B5 respectively. Compared to the baseline, it was noted that NaHS registered a ,50% suppression of I K in hESC-CM (A1) and hiPSC-CM (B1). Such effect of NaHS was equivalent to the inhibitory effects on I K (44.662.6 and 47.763.7) achieved by combined Chromanol 293B and E-4031 which blocked both I Ks and I Kr in hESC-CMs (A2) and hiPSC-CMs (B2). After subtracting the effects of NaHS from the effects of both I K blockers, additional treatment with NaHS did not show a significant further inhibition on I K (A2 and B2), suggesting that the effect of NaHS overlapped with those of Chromanol 293B and E-4031 blockers. Moreover, it was noted that NaHS exerted additive inhibitory effect on total I K in the respective presence of inhibitory effects of Chromanol 293B on I Ks (A3 and B3) and E-4031 on I Kr (A4 and B4), suggesting that NaHS suppressed both I Ks and I Kr in both hESC-CMs and hiPSC-CMs. However, the effects of NaHS on A-like CMs were inconclusive due to insufficient number of cells (n ,4 for each subgroup) tested. No effects of NaHS on N-like CMs were observed (data not shown).
Exposure to NaHS Significantly Inhibited L-type Ca 2+ Current in H9 hESC-CMs Ca 2+ signaling plays a crucial role in cardiac excitationcontraction (EC) coupling. L-type Ca 2+ channel is responsible for Ca 2+ influx triggered by the electrical signal during cardiac contraction. I CaL contributes to the 2 nd phase of action potential and is more prominent in ventricular cardiomyocytes. Cardiomyocytes exposed to NaHS showed a decreased I CaL density. Fig. 5A shows that the I CaL density was significantly decreased in H9 hESC-derived V-like CMs exposed to 300 mM NaHS from 21562.9 pA/pF to 21063.2 pA/pF at 0 mV (p,0.05). The specificity of I CaL was confirmed by 20 mM verapamil, a blocker of I CaL which expectedly reduced the current substantially (p,0.05), while the addition of 300 mM NaHS in the presence of 20 mM verapamil failed to see a further reduction of I CaL , suggesting that the effect of NaHS overlapped with verapamil (Fig. 5B). It thus further confirmed the effect of NaHS on I CaL . Furthermore, the I-V relationships following NaHS treatment suggested a concentration-response curve of I CaL inhibition by NaHS (Fig. 5C).

Exposure to NaHS Significantly Inhibited I f Current in hiPSC-derived CMs
Interestingly, I f current was detected in hiPSC-derived V-like CMs which was significantly reduced by addition of specific channel blocker, CsCl (Fig. 7A). Similarly, exposure to NaHS (100 mM) significantly decreased I f current in those CMs (p,0.05) (Fig. 7Bd).

Discussion
Hydrogen sulfide (H 2 S) as a recently identified gaseous signalling transmitter has been known to interact with a wide range of ion channels to mediate important physiological responses. These include protecting against myocardial ischemic reperfusion injury and other cardiac protective effects through modulation of ATP-sensitive potassium current (I kATP ) [17] and voltage-gated L-type calcium current (I CaL ) [18]. Despite its beneficial effects, H 2 S may have implication in cardiac arrhythmias as it interacts with multiple ion channels involved in membrane action potential [7].
Outward rectifier potassium currents (I K ) including slow and rapid I K (I Ks and I Kr ) contribute to the 2 nd and 3 rd phase of AP, and both play an important role in regulating the repolarization of cardiomyocytes. Drugs affecting I Ks and I Kr have been associated with cardiac arrhythmia [1,2]. Both I Ks and I Kr are the predominant I K in adult human CMs [19] and their presence has been confirmed in hiPSC-CMs [10,20,21]. To our knowledge, the findings of inhibitory effect on delayed rectifier I k of human CMs is the first report of such effect by H 2 S. Our data showed that H 2 S at physiological concentration could suppress both I Ks and I Kr currents. In addition, H 2 S was found to mediate I CaL channel inhibition in V-like CMs derived from H9 hESCs. This is consistent with previous report that H 2 S inhibited I CaL in rat Vlike cardiomyocytes [22].
In human CMs, the outwardly rectifying potassium current I Ks and I Kr contribute to phase 2 and 3 repolarization of AP in both V-and A-CMs [23]. In V-CMs, the longer phase 2 of AP of membrane depolarization is contributed mainly by I CaL . In the present study, the suppressed I Ks and I Kr by H 2 S contributed to prolonged repolarization phase in V-like and A-like CMs with appreciable perturbation of APD. In V-like CMs, however, altered APD could be the consequence of a balanced inhibitory effect of H 2 S on delayed rectifier I K and on I CaL . Although suppressing I CaL is likely to shorten the APD, inhibiting outwardly rectifying potassium current in phase 2 and phase 3 by H 2 S may override such effect, resulting in an overall APD prolongation as observed in our study. In contrast to V-and A-like CMs, N-like CMs derived from H9 hESC and hiPSCs did not show significant difference in APD in response to H 2 S.
In contrast to adult CMs of which only nodal CMs are capable of spontaneous contraction, all CMs derived from hiPSCs are capable of spontaneous contraction [10] indicating their fetal-like phenotype in culture. The identification of I f current in hiPSCderived V-like CMs in this study further supports the immature status of those hiPSC-CMs. Nevertheless, inhibition of I f may explain the inhibitory effect of H 2 S on the contraction rates of hiPSC-CMs observed. However, since sulfhydration of K ATP by H 2 S has been attributed to its channel opening effect [24,25], it will be interesting to study if S-sulfhydration by H 2 S had any effect on I K , I CaL and I f channel activities in the future. Furthermore, endogenous H 2 S mediated by cystathionine-b-synthase on the observed outcomes were not studied, though it has been shown to have effect similar to I KATP channel opening effect of exogenously supplied NaHS in rat myocytes [26].
In conclusion, we present the first report of inhibition of both slow and rapid delayed rectifier potassium channels and hyperpolarization-activated inward current in human cardiomyocytes by H 2 S. Such effect, in combination with its inhibition of I CaL , could contribute to prolonged APD and slowed contracting rates in V-and A-like CMs that may have undesired implications in its vasodilation function in cardiac hemodynamics.