Gram‐negative bacteria‐induced infections result in fever, arrhythmia, and even death. Lipopolysaccharide (LPS), a constituent of bacteria, leads to an inflammatory response under sepsis and increase arrhythmogenesis. This study analyzed the effects on human embryonic stem cell‐differentiated cardiomyocytes (HIPSC‐CMs) exposed to LPS. A whole cell patch clamp was used to record the action potential (AP) and ionic currents with or without different doses of LPS in HIPSC‐CMs. Compared with the control, a different dose (0.04, 0.2, 1, and 5 µg/ml) of LPS‐treated HIPSC‐CMs resulted in a longer AP duration. The IC50 of sodium channel current was 1.254 µg/ml, L‐type calcium channel current was 5 µg/ml, and I_k channel currents were 1.254 µg/ml. LPS‐treated HIPSC‐CMs showed a lower sodium channel current, L‐type calcium channel current, and I_k channel currents. Furthermore, the expressions of Nav1.5 were decreased, and L‐Ca, Kv11.1, and Kv7.1 were increased in LPS‐treated HIPSC‐CMs. LPS‐induced arrhythmogenesis was related to the electrophysiological characteristics of sodium channel current, L‐type calcium channel current, and I_k channel currents. These results suggest a potential mechanism of cardiomyocyte injury in endotoxemia.

2.1. Ethics statement

All of the procedures conformed to the NIH guidelines and were conducted following the protocols approved by the Animal Care Committee of Inner Mongolia University for Nationalities.

2.2. Generation of HIPSC‐CMs

Human embryonic stem cells were cultured and differentiated into HIPSC‐CMs according to previous research (Lu et al., 2014). The human embryonic stem cells were seeded into low adhesion six‐well plates in suspension culture to form embryoid bodies. After 7 days, the embryogenic bodies were inoculated into four‐well plates and adhered to the wells with the final concentration of 10 µmol/L 5‐azacytidine as differentiation induction. HIPSC‐CMs were kept at 37°C in a water vapor‐saturated atmosphere with 5% CO_2, and they were used from 24 hr up to 6 weeks after passaging with a medium change every 48 hr. The HIPSC‐CMs were divided into the following groups: the control group and the LPS group (0.04, 0.2, 1, and 5 ug/ml).

For whole cell patch clamp experiments, HIPSC‐CMs were incubated with collagenase CLSI for 30 min at 37°C, and then added to an Roswell Park Memorial Institute (RPMI) medium containing 10 FCS (Worthington, Germany) to collect HIPSC‐CMs.

2.3. Whole cell patch clamp experiments

The AP and ion channel currents were measured by standard patch clamp recording. Digidata 1440A, multiclamp 700B, micromotor drawing instrument MODEL P‐97, and three‐dimensional manipulator MP‐225 (Sutter Medical Technologies, Freiburg, Germany) were used for this study.

Patch electrodes were pulled from borosilicate glass capillaries using a DMZ‐Universal Puller. Patch electrodes were filled with electrode liquid. Before a Giga‐seal was formed, electrode offset potentials were zero‐adjusted. Fast capacitance was first compensated, and then the membrane under the pipette tip was disrupted by negative pressure after a Giga‐seal was generated to establish the whole cell configuration. The potential was recorded in a current‐clamp mode, and ion currents were analyzed in the voltage‐clamp mode.

The sodium channel current was recorded during depolarization, with the holding potential of −90 mV and a test potential −20 mV at room temperature. The current–voltage (I–V) curve of I _Na was obtained by plotting the I _Na peak current density at each test potential as the ordinate and the detection voltage as the abscissa. An external solution containing (mmol/L) choline chloride 110.0, NaCl 30.0, KCl 5.4, MgCl₂ 1.0, HEPES 10.0, Glucose 10.0, tetraethylammonium (TEA) 10.0, 4‐AP 1.0, CdCl₂ 0.2, and NiCl₂ 0.1, pH 7.4 was used. The intracellular solution consisted of (mmol/L) CsCl 140.0, CaCl₂ 1.0, MgCl₂ 2.0, HEPES 10.0, EGTA 10.0, and Mg‐ATP 5.0, at pH 7.2.

The I _Ca‐L channel current was recorded during depolarization, maintained at a potential of −40 mV, to testing potentials +20 mV at a room temperature, and was then restored back to −40 mV. The recorded I _Ca‐L was fitted by the Boltzmann equation: I/I _max = 1 / {1 + exp [(V – V1 / 2) / k]}, where I and I _max are the current and maximum current at different test voltages Current, V denotes the pulse voltage, V1 / 2 denotes the pulse voltage of 50% of the channel inactivation, and k denotes the slope factor. An external solution containing (mmol/L) TEA 135, MgCl₂ 0.53, CaCl₂1.8, CsCl 20, and HEPES 5 at a pH of 7.4, and an intracellular solution consisting of (mmol/L) CsO H110, aspartic acid 90, CsCl 20, TEA‐Cl 10, HEPES 10, EGTA 10, Mg‐adenosine triphosphate (Mg‐ATP) 5, Na₂ creatine phosphate 5, guanosine triphosphate (GTP) (Tris) 0.4, and leupeptin 0.1, at a pH of 7.2 was used.

The I _ks channel current was recorded during depolarization, from a holding potential of −80 mV to testing potentials 40 mV to −40 mV at room temperature. An external solution containing (mmol/L) 135 NaCl, 5 KCl, 1 MgCl₂, 2.8 Na Acetate, and 10 HEPES, was used, and the pH was adjusted to 7.4 with NaOH. The intracellular pipette solution contained (mmol/L) 130 KCl, 5 EGTA, 1 MgCl₂, 4 Na₂‐ATP, 0.1 GTP, 10 HEPES, with the pH adjusted to 7.2 with KOH.

hERG K+ currents were recorded under a holding potential at −60 mV and then depolarized from −60 mV to 60 mV stepped by 10 mV for 4 s to activate the hERG K+ channel, then the peak tail currents were induced by a repolarizing pulse to −40 mV for 4 s. The bath solution contained 126 Choline‐Cl, 5 KCl, 2CaCl₂, 1 MgCl₂, 10 HEPES, and 5 glucose, with the pH adjusted to 7.4 with Choline‐OH. The solution was supplemented by 4‐AP (5 mmol/L), BaCl₂ (0.5 mmol/L), CdCl₂ (0.2 mmol/L), and chromanol 293B (10 μM) to suppress the potential interference of INa, Ito 1, IK1, ICaL, and IKs, respectively. A pipette solution containing 20 mmol/L KCl, 110 mmol/L K‐Aspartic, 1 mmol/L MgCl₂, 5 mmol/L EGTA, 10 mmol/L HEPES, and 2 mmol/L Na₂‐ATP was used, and the pH was adjusted to 7.2 with KOH.

2.4. Western blot analysis

Protein lysates were separated by Sodium Dodecyl Sulfate‐polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes, and incubated overnight with primary antibodies. The following concentration of primary antibodies was used (anti‐Kv7.1 1:200, anti‐Nav1.5 1:1000, anti‐Kv11.1 1:1000 [OriGene, Rockville, MD]; anti‐cTnT 1:1000, anti‐α‐actinin 1:1000, and anti‐calcium channel L‐type DHPR alpha 2 subunit 1:1000 Abcam, Cambridge, UK). Signals were detected and quantified by chemiluminescence. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as the loading control.

2.5. Statistical analysis

All of the values are expressed as mean ± standard error of the mean. SSPS 17.0 statistical analysis software was used for data analysis. A paired Student’s t test was used to compare the different groups. The number of stars (*/#) indicate the p value range: *p value < 0.05, **p value < 0.01, ***p value < 0.001; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001.

3.1. LPS induced tachycardia in HIPSC‐CMs

HIPSC‐CMs were analyzed by their characteristic cardiac‐specific proteins and AP traces. There was low expression of cardiac troponin‐T (cTnT) and α‐actinin, whereas the expressions of cTnT and α‐actinin were significantly increased on the ninth day of differentiation (Figure ​(Figure1).1). It showed the typical AP trace of HIPSC‐CMs paced. The different doses (0,04, 0.2, and 1 μg/ml) of LPS were used to treat HIPSC‐CMs to evaluate the effects of LPS on the AP morphology. A significant difference was observed in APD among the control and LPS (0.04, 0.2, and 1 μg/ml) groups. LPS‐treated HIPSC‐CMs showed a dose‐dependent lengthening of the APD when compared with the control (Figure ​(Figure2).2). Moreover, to analyze the effect of LPS on the spontaneous AP, HIPSC‐CMs were treated with 1 μg/ml LPS. Compared with the control, LPS treatment induced tachycardia in HIPSC‐CMs. Tachycardia dissipated after the washing of LPS (Figure ​(Figure33).

Figure 1

The characteristics of HIPSC‐CMs were analyzed by cardiac‐specific proteins. (a) Expression of cTnT in HIPSC‐CMs was assessed by western blot analysis. (b) Western blot analysis analysis of α‐actinin expression in HIPSC‐CMs. **p < 0.01 vs Con. cTnT: cardiac troponin‐T; HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide

Figure 2

Effect of LPS on the spontaneous AP of HIPSC‐CMs. AP: action potential; HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Effect of LPS (1 μg/ml) on the spontaneous AP of HIPSC‐CMs. (a) Representative recording showing the spontaneous AP before LPS application. (b) Decreased beating rate and prolonged AP duration after LPS (1 μg/ml) application. (c) Recovery from tachycardia after washout of LPS. Spontaneous AP under current clamp configuration and the membrane current was held at 0 mV. AP: action potential; HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide

3.2. LPS reduced L‐type calcium current in HIPSC‐CMs

To investigate the ion channel currents that may contribute to the LPS‐induced change of APD, L‐type calcium current was analyzed in HIPSC‐CMs. LPS demonstrated weaker state‐dependent inhibition of the sodium channel current while dose‐dependently inhibiting inactivated L‐type calcium channel current (Figure ​(Figure4a)4a) with an IC50 of 1.254 µg/ml (Figure ​(Figure4).4). An I–V curve was made with pulse voltage as abscissa, corresponding to the current density (pF/pA) as the vertical axis. LPS significantly inhibited I _Ca‐L current amplitude (pA/pF), demonstrating a 41% decrease in the peak current, but the I _Ca‐L voltage‐dependent activation and steady‐state inactivation were not statistically significant (Figure ​(Figure44).

Figure 4

Effect of LPS on L‐type calcium channel currents in HIPSC‐CMs. (a) Representative traces of I _Ca‐L at +20 mV. (b) The inhibition of the I _Ca‐L current was analyzed by different doses of LPS. (c) The IC50 of I _Ca‐L was analyzed. (d) Representative activation curves of peak I _Ca‐L. (e) Representative inactivation curves of peak I _Ca‐L. (f) Mean values of Ca peak current at +20 mV. *p < 0.05 vs Con. HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide

3.3. LPS reduced sodium channel currents (I _Na) in HIPSC‐CMs

The effect of LPS in prolonging the APD suggested that sodium channel currents (I _Na) might be affected by LPS. The effects of LPS on I _Na were examined. LPS demonstrated strong state‐dependent inhibition of sodium channel currents while dose‐dependently inhibiting inactivated sodium channel currents (Figure ​(Figure5a)5a) with an IC50 of 1.254 µg/ml (Figure ​(Figure55).

Figure 5

Effect of LPS on sodium channel currents in HIPSC‐CMs. (a) Representative traces of I _Na at −20 mV in the absence and presence of 20 µM TTX. (b) The inhibition of sodium channel currents was analyzed by different doses of LPS. (c) The IC50 of the sodium channel currents was analyzed. (d) Representative activation curves of peak I _Na. (e) Representative inactivation curves of peak I _Na. (f) Mean values of Na at –20 mV. *p < 0.05 vs Con. HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide; TTX: Tetrodotoxin

Compared with the control group, LPS decreased the amplitude of I _Na at all of the tested potentials; all five doses of LPS significantly reduced the inward peak amplitude of I _Na and I _Na current density, with a 61% decrease in the peak current. Next, the voltage‐dependent activation and steady‐state inactivation were evaluated by plotting Na⁺ peak current. The voltage‐dependent activation curve of I _Na was shifted left in HIPSC‐CMs with LPS (Figure ​(Figure5d),5d), and the steady‐state inactivation curve of I _Na in HIPSC‐CMs with LPS was shifted left (Figure ​(Figure55e).

3.4. LPS reduced I_k channel currents in HIPSC‐CMs

In human cardiomyocytes, outward rectifier potassium currents (I _k) play a critical role in cardiac AP repolarization. I _k (I _ks and I _kr) currents were activated and recorded. I _ks was quantified by fitting the Hill equation: I _compound / I _control = 1 /[1 + (the concentration of LPS / IC50)ⁿ], where IC50 is the drug concentration for 50% inhibition, and n is the Hill coefficient. Cultured HIPSC‐CMs were exposed to LPS at 0.04, 0.2, 1, and 5 μg/ml and I _ks was recorded 3 min later. The IC50 was 1.254 µg/ml. Furthermore, LPS shifted the half‐activation voltage of I _ks in HIPSC‐CMs with a 54% decrease in the peak current, and the slope factor significantly differed (Figure ​(Figure6).6). LPS increased I _kr significantly (Figure ​(Figure77).

Figure 6

Effect of LPS on slowly delayed rectifier currents (I _Ks) in HIPSC‐CMs. (a) The I _ks channel current was recorded during depolarization, from a holding potential of −80 mV to testing potentials 40 mV to −40 mV at a room temperature. (b) Mean values of I _Ks at +40 mV. (c) The IC50 of I _Ks was analyzed. (d) Mean values of I _Ks at +60 mV. *p < 0.05 vs Con. HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide

Figure 7

Effect of LPS on rapidly delayed rectifier currents (I _Kr) in HIPSC‐CMs. (a and b) hERG K + currents were recorded under a holding potential at −60 mV and then depolarized from −60 mV to 60 mV stepped by 10 mV for 4 s to activate the hERG K + channel, then the peak tail currents were induced by a repolarizing pulse to −40 mV for 4 s. (c and d) The tail current and prepulse current of I _Kr were analyzed. (e) Mean values of hERG tail current (prepulse at +60 mV). HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; hERG: human ether‐a go‐go‐related gene; LPS: lipopolysaccharide

3.5. LPS changed the expression of ion channels

HIPSC‐CMs were treated for 24 hr with 1 μg/ml LPS. Protein expression of ion channels was analyzed by western blot analysis. LPS reduced the expression of Nav1.5 (Na⁺ channel, Figure ​Figure8a),8a), but enhanced the expression of I _CaL (Figure ​(Figure8b),8b), Kv7.1 (I _Ks, Figure ​Figure8c),8c), and Kv11.1 (I _Kr, Figure ​Figure88d).

Figure 8

LPS induced changes in protein expression of ion channels. (a–d) The expression of Nav1.5/ICaL/Kv7.1 /Kv11.1 were analyzed by western blot analysis in HIPSC‐CMs after treatment by LPS. *p < 0.05, **p < 0.01 vs Con. HIPSC‐CMs: human embryonic stem cell‐differentiated cardiomyocytes; LPS: lipopolysaccharide

We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.

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