Effects of Sleep Deprivation on Action Potential and Transient Outward Potassium Current in Ventricular Myocytes in Rats

Background

Sleep disorder is a growing problem in modern society that affects life quality and is becoming a major health problem [1]. Both acute and chronic sleep restriction have adverse effects on cardiovascular system, immune responses, hormonal pathways, and thermoregulation. Human studies show that sleep deprivation can increase activation of the autonomic nervous system (ANS), the hypothalamic-pituitary-adrenal axis, and the immune system [2–6]. Sgoifo et al. reported that the heart rate and hypothalamic-pituitary-adrenocortical (HPA) axis activity significantly increased after 48-h sleep deprivation [1]. Some studies reported that electrocardiographic changes result from parasympathetic and sympathetic tones imbalance caused by sleep deprivation. Both in healthy young adults and in rat models, it had been documented that acute sleep deprivation was associated with the increase of QT interval and QT dispersion, which suggests that sleep deprivation may increase the risk of ventricular arrhythmia [7,8]. It had been known that the prolongation of the action potential duration (APD) can result in the increase of the QT interval and QT dispersion. Change of transient outward current (Ito) is an important ionic mechanism that contributes to this process in several pathophysiologic conditions [9,10]. However, little is known about the effects of sleep deprivation on the ion channels, such as Ito of ventricular myocytes. The present study was designed to investigate the effects of sleep deprivation on the APD and Ito of rat ventricular myocytes.

Material and Methods

ANIMALS:

Fifty Sprague-Dawley adult male rats (200–250 g) were housed in cages with a 12-h: 12-h light-dark cycle, and the room temperature was controlled at 22–24°C. Before sleep deprivation, rats were allowed to stay in the same cage for 2 weeks to establish a social hierarchy within the group. All procedures of this study complied with the Guide for the Care and Use of Laboratory Animals. The rats were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. and the certificate no. was SCXK (Jing) 2012-0009.

EXPERIMENTAL PROCEDURE:

The same tank used for the sleep deprivation group was used for the platform control group, but a platform (30.0×18.0 cm) was placed in cage, and rats had certain activities and could sleep on the platform. For the SD groups, rats were divided into groups based on the duration of SD: 1, 3, 5, and 7 days (10 rats for each duration). Methods for sleep deprivation were that rats were group-housed (5 rats in each arena) in modified multiple platform arenas during SD. The experimental group was submitted to SD using the modified multiple platform method. Briefly, rats were placed in an acrylic water tank (70×50×40 cm) containing 5 circular platforms, 6.3 cm in diameter, with water filled to 1 cm beneath the surface of platforms. The rats could move around inside the tank by jumping from 1 platform to another. This method relies on the muscle atonia that accompanies paradoxical sleep. When the rats on the platforms reach this sleep stage, they lose muscle tone; therefore, they either touch or fall into the surrounding water, so they are awakened. The modified multiple platform method completely abolishes paradoxical sleep and also decreases slow wave sleep by approximately 35% [11]. Food and water were provided by placing food pellets and water bottles on a grid located on top of the tank. The water in the tank was changed daily throughout the SD period.

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Tyrode’s solution contained (mM/L): NaCl 126, KCl 5.4, MgCl₂ 1, CaCl₂ 1.8, NaH₂PO₄ 0.33, glucose 10 and 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, pH adjusted to 7.4 with NaOH. The Ca²⁺-free Tyrode’s solution was prepared by removing CaCl₂ from the Tyrode’s solution.

The enzyme solution used for the rat cardiomyocyte isolation contained 0.1g/L collagenase (type II) and 0.5g/L BSA in Tyrode’s solution.

For recording I_to currents, the pipettes were filled with (mM/L): K-aspartame acid 140, MgATP 4, MgCl₂ 1, Ethylene glycol tetraacetic acid (EGTA) 10, Guanosine triphosphate (GTP) 0.1, HEPES 10, pH adjusted to 7.3 with KOH.

For recording I_to currents, myocytes were superfused with solution containing (mM/L): NaCl 140, KCl 4, CaCl₂ 1, MgCl₂ 1, HEPES 10, glucose 5, pH adjusted to 7.4 with NaOH. Tetrodotoxin inhibits Na⁺ current; CdCl₂ inhibits Ca²⁺ current.

CELL PREPARATIONS:

After SD, rats were given intraperitoneal injection of pentobarbital sodium (100 mg/kg) and a single ventricular myocyte was dissociated from excised perfused hearts by an enzymatic dissociation as previously described [12]. Briefly, hearts were excised rapidly and retrogradely perfused on a Langendorff apparatus with a Ca2+-free Tyrode’s solution for 5 min before the perfusate was switched to an enzymatic solution for 15 min. The perfusates were bubbled with 95% O2 +5% CO2 and maintained at 37°C. The ventricles were cut into small chunks and gently agitated in Ca2+-free Tyrode’s solution. The cells were filtered through nylon mesh (pore size 200 μm) and stored in Ca2+-free Tyrode’s solution at 4°C.

ELECTROPHYSIOLOGICAL MEASUREMENTS:

Patch clamp experiments were performed on isolated ventricular cardiomyocytes. Quiescent, calcium-tolerant, rod-shaped cells with clear cross striation were used for action potential recordings at 35°C. Transmembrane potentials and currents were recorded using the whole cell patch-clamp technique with a MultiClamp 700B amplifier (Axon Instruments). All signals were acquired at 5 kHz (Digidata 1322A, Axon Instruments) and analyzed by pCLAMP version 9.2 software (Axon Instruments). Whole-cell currents and Action potentials (APs), obtained under voltage clamp, were filtered at 1–5 kHz and sampled at 5–50 kHz, and the series resistance was typically <5 mega ohms after about 70% compensation. The P/4 protocol was used to subtract online the leak and capacitive transients.

APs were elicited using the current-clamp mode at a rate of 5.0 Hz of 30 train suprathreshold current pulses. Cardiomyocytes were electrically stimulated by intracellular current injection through patch electrodes using depolarizing pulses with a duration of 5 ms and an amplitude of 1500 pA. Action potential duration (APD) was measured at 20% and 50% of repolarization (APD₂₀ and APD₅₀). I_to was recorded using the voltage-clamp mode. We used pre-pulse to −40 mV for 30 ms to inactivate I_Na. I_to was recorded in voltage-clamp mode with 300-ms pulses from a holding potential of −80 mV, with different test potentials increased from −40 mV to +70 mV with 10-mV steps. Steady-state activation of I_to was induced by voltage steps between −60 mV and +40 mV for 500 ms from a holding potential of −80 mV.

Steady-state inactivation of I_to was induced by a condition pulse of +40 mV for 20 ms, following voltage steps between −120 mV to +30 mV, with 10 mV for 1000 ms. Voltage-dependence of the time course of recovery from inactivation was evaluated with a paired-pulse protocol: conditioning pulse was applied to +40 mV for 300 ms from holding potential of −80 mV, following test potentials of +40 mV for 300 ms during different time intervals of 20 ms,40 ms,80 ms, 160 ms, 320 ms, 640 ms, and 920 ms. The time course of recovery from fast inactivation was fitted by single-exponential function. The time constant of closed-state inactivation of I_to was induced by following depolarization pulse of +50mV for 300 ms return to −100 mV, depolarizing to −70 mV during different time pluses of 10 ms, 20 ms, 50 ms, 100 ms, 200 ms, 500 ms, 1000 ms, 2000 ms, 2500 ms, 4000 ms and 5000 ms. Time constant of inactivation was fitted by single-exponential function.

STATISTICAL ANALYSIS:

The data are presented as Mean ±SD. The curves were fitted with pCLAMP 10.0 (Axon Instruments) and software Origin 6.0. The statistical significance was determined using ANOVA to compare multiple groups. A value of P<0.05 was considered statistically significant.

Results

EFFECTS OF SLEEP DEPRIVATION ON APD:

Action potential traces were recorded in sleep deprivation 1, 3, 5, and 7 days groups and compared with the control group. Figure 1 showed that the action potential duration was prolonged after 3 days of sleep deprivation. The APD20 values of the 3, 5, and 7 days SD groups (8.28±0.30 ms, 11.56±0.32 ms and 13.24±0.56 ms), were longer than the 1 day SD group (5.77±0.20 ms) and control group (5.66±0.16 ms, P<0.01, n=18). The APD50 values of the 3, 5, and 7 days SD groups changed from 52.77±3.20 ms (1 day group), to 65.28±5.30 ms, 83.56±7.32 ms, and 89.24±5.56 ms. These results demonstrated that both the APD20 and APD50 were prolonged after sleep deprivation (Table 1).

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In Figure 2A, the left inset shows Ito current traces obtained by applying train pulses. Current amplitudes of 3, 5, 7, days SD groups were markedly smaller than the 1 day SD group and control group. At test potentials of +70 mV, current densities of Ito were 39.84±3.01 pA/pF in the 1 day group, 27.38±2.42pA/pF in the 3 days group, 21.38±1.47 pA/pF in the 5 days group, 13.74±0.98 pA/pF in the 7 days group, and 40.60±4.04 pA/pF in the control group (P<0.01,n=18, Figure 2B). After sleep deprivation, the outwardly rectifying of Ito was reduced or even disappeared. In the 7 days group, the current density-voltage (I–V) curve was almost a straight line. The current-voltage relationship showed slower acceleration of densities (Ito) in the 3, 5, and 7 days group and a more positive membrane potential of +10 mV (Figure 2C).

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The changes in the Ito gating mechanism associated with SD have also been studied. After sleep deprivation, the steady-state activated curve was shifted to positive potentials (Figure 3A–3C). The half-activation potential (V1/2,act) was −31.51±1.75 mV in the control group, −22.62±0.53 mV in the 1 day group, −14.47±0.86 mV in the 3 days group, −11.58±0.47 mV in the 5 days group, and −0.98±0.66mV in the 7 days group (P<0.01, n=18). The slope factors of activation curve (kact) did not change significantly in any group.

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After sleep deprivation, the steady-state inactivated curve was shifted to left and the half-inactivation potentials (V1/2,inact) of Ito were shifted towards depolarization. There were no changes in the slope factors of the inactivation curve (kinact) (Figure 3D–3F).

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A slow recovery from inactivation of the Ito in sleep deprivation cardiomyocytes was obtained. Figure 4A shows that the current recovery of inactivation was slow after sleep deprivation. When recovery time was shortened to 100 ms, this phenomenon was more obvious (Figure 4B).

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The inactive, closed-state of Ito currents were accelerated in cardiomyocytes from sleep deprivation rats. At the 5000 ms, about 95.1% Ito channels were opening in cardiomyocytes of the control group, but only 80.6–84.9% of Ito channels were opening after sleep deprivation (Figure 5).

Discussion

LIMITATIONS:

In the present study, we only focused on the changes of Ito current density and its impact on the action potential configuration after sleep deprivation. Sallé et al. suggested that the Ca2+ current was also involved in the lengthening of APD [51]. We plan to investigate Ca2+ current changes after SD in our future work. Additionally, we evaluated the electrophysiologic changes of ventricular myocytes after acute sleep deprivation in this study, but more work is needed in the field of chronic sleep deprivation.

Conclusions

Our study shows that decreased activation and accelerated inactivation of the Ito channel caused the reduction of Ito current density after sleep deprivation, which resulted in prolonged APD of ventricular myocytes.