Effect of iron overload on electrophysiology of slow reaction autorhythmic cells of left ventricular outflow tract in guinea pigs

Ling Fan, Li-Feng Chen, Jing Fan, Lan-Ping Zhao, Xiao-Yun Zhang

1Department of haematology, the First Affiliated Hospital of Hebei North University, 075000 Zhangjiakou, Hebei, China

2Department of Physiology, Basic Medical College, Hebei North University, 075000 Zhangjiakou, Hebei, China

3Department of Obstetrics and Gynecology, the Sixth Hospital of Zhangjiakou, 075000 Zhangjiakou, Hebei, China

1. Introduction

In clinic, there are some diseases such as thalassemia, aplastic anemia and myelodysplastic syndrome, which require long-term blood transfusion. Iron overload caused by blood transfusion can lead to myocardial injury and cause arrhythmia[1-4]. Our previous study found that the slow response autorhythmic cells in the ventricular outflow tract were closely related to the ventricular arrhythmia[5,6]. There was no report of the electrophysiological effects and its mechanisms of iron overload on the autorhythmic cells in the ventricular outflow tract. In this experiment, exogenous iron was applied to the left ventricular outflow tract tissue to simulate the electrophysiological effects of iron poisoning on the slow response autorhythmic cells in order to explore the mechanism of ventricular arrhythmia induced by iron overload.

2. Materials and methods

2.1. Experimental animals and reagents

A total of 64 guinea pigs comprising both males and females,weighing 250-350 g, were purchased from the Beijing Jinmuyang Experimental Animal Breeding Co. Ltd. [(animal license No.:SCXK (Beijing) 2010- 0001)]. The guinea pigs were randomly divided into eight groups as following: control group (n= 8), FeSO4(100 μmol/ L) group (n= 8), FeSO4(200 μmol/L) group (n= 8),CaCl2(4.2 mmol/L)+FeSO4(200 μmol/L) group (n= 8), Verapamil(1 μmol/L) group (n= 8), Verapamil (1 μmol/L)+ FeSO4(200 μmol/L) group (n= 8), nickel chloride (200 μmol/L) group (n= 8), and nickel chloride (200 μmol/L)+FeSO4(200 μmol/L) group (n= 8).FeSO4·7H2O was produced by Tianjin Dingshengxin Chemical Co.Ltd. Verapamil by BioMol company. Nickel chloride (NiCl2·7H2O)by Tianjin Kaixin Chemical Industry Co. Ltd.

2.2. Preparation of specimen

After the guinea pigs were anesthetized with ethyl carbamate, their chest were cut open and hearts removed. The hearts were quickly placed in O2saturated modified Locke solution. The tissue specimen of the left ventricular outflow tract was made[7-9] and fixed with stainless steel needle on the silicone rubber in the perfusion chamber(1.5 cm × 2 cm, the volume is about 4 mL). The O2saturated modified Locke solution (NaCl 157 mmol/L, KCl 5.6 mmol/L,CaCl22.1 mmol/L, NaHCO31.8 mmol/L, glucose 5.6 mmol/L, and PH7.3 to 7.4) were perfused at constant temperature of (35±1) ℃and constant speed 10 mL/min. Animal breeding, care and all experiment porcedures were performed in adherence to guidelines by Hebei North University animal experiment center and approved by Animal Ethics Committee.

2.3. Potential guidance

After the glass microelectrode was filled with the saturated KCl electrode solution, the DC resistance is 10-20 MΩ. The electrode was inserted into the lower part near the middle of the right and posterior flap. In most cases, the spontaneous slow action potential could be recorded directly by this way; otherwise the stimulation electrodes were placed at the heart tissue away from the valve in square wave stimulation of 2 ms, 1 Hz, and twice threshold intensity(YC-2 stimulator by Chengdu Instrument Factory); stimulation time ranged from a few seconds to a few minutes until a stable induced spontaneous rhythm appearing, stop stimulation began the experiment. The spontaneous slow reaction potential was amplified by SWF-1B type microelectrode amplifier (Chengdu instrument factory), and the results were input into a microcomputer by RM6280C multi-channel physiological signal acquisition system(Chengdu Instrument Factory) to display electrical signals, and the parameters of spontaneous slow response potential to be analyzed.

2.4. Index observation

The following indexed were observed: Maximal diastolic potential(MDP), amplitude of action potential(APA), maximal rate of depolarization (Vmax), velocity of diastolic depolarization(VDD),rate of pacemaker firing(RPF), 50% and 90% of duration of action potential (APD50and APD90).

2.5. Experimental process

Perfusion was conducted with the saturated O2modified Locke liquid, and when the spontaneous rhythm stabilized for 20 min, a set of normal slow spontaneous potential was collected as control group.Then, perfusion was conducted with drug of different concentrations and saturated O2modified Locke liquid. Potential changes were recorded at 0.5, 1, 2, 5 min, respectively after each perfusion. After each observation of the drug effect, the modified Locke solution saturated with O2was applied to washout for 20 min in order to observe the recovery of spontaneous slow reaction potential.

2.6. Experimental scheme

2.6.1. The electrophysiological effects of Fe2+ on the Slow reaction autorhythmic cells of the left ventricular outflow tract

Left ventricular outflow tract tissue was perfused with modified Locke solution containing FeSO4(100 μmol/L) and FeSO4(200 μmol/L), respectively to observe different concentrations of FeSO4on the electrophysiological changes of the slow reaction autorhythmic cells of the left ventricular outflow tract.

2.6.2. Effects of improved Ca2+ concentration in perfusion fluid on the electrophysiological effect of FeSO4 (200 µmol/L)

The left ventricular outflow tract tissue was perfused with Locke solution, in which doubled concentration of Ca2+(CaCl2, 4.2 mmol/L) and FeSO4(200 μmol/L) were added to observe the influences of high concentration of Ca2+on electrophysiology of FeSO4(200 μmol/L).

2.6.3. Verapamil’s effects on electrophysiological effect of FeSO4 (200 µmol/L)

The left ventricular outflow tract tissue was perfused with Locke solution containing Verapamil (1 μmol/L) and FeSO4(200 μmol/L) in order to observe the effects of Verapamilon the electrophysiological effects of FeSO4(200 μmol/L).

2.6.4. Effects of nickel chloride on electrophysiological effects of FeSO4 (200 µmol/L)

The left ventricular outflow tract tissue was perfused with Locke solution containing nickel chloride (200 μmol/L) and FeSO4(200 μmol/L) to observe the influences of nickel chloride on the electrophysiological effects of Fe2+(200 μ mol/L).

2.7. Statistical analysis

Software SPSS version 16.0 was used for statistical analysis.Measurement data was expressed as Mean ± SD. Self pairedttest was used for comparison of different indexes before and after treatment.P＜0.05 was considered as significant difference.

3. Results

3.1 The electrophysiological effects of Fe2+ on the Slow reaction autorhythmic cells of the left ventricular outflow tract

FeSO4(100, 200 μmol/L) can change the electrophysiologgical indexes of the slow response autorhythmic cells of the left ventricular outflow tract in a concentration-dependent manner, make Vmax,APA and MDP decreased, RPF and VDD slowed, APD50and APD90prolonged (P＜0.05) (Table 1).

3.2. Influences of high concentration of Ca2+ on the electrophysiological effects of FeSO4 (200 µmol/L)

Increased concentration of Ca2+(CaCl2, 4.2 mmol/L) could partially inhibit the electrophysiological effects of FeSO4(200 μmol/L) (Table 2).

3.3. Verapamil’s effects on electrophysiological effect of FeSO4 (200 µmol/L)

The LTCC channel blocker Verapamil (1 μmol/L) could block the electrophysiological effects of FeSO4(200 μmol/L) (Table 3).

3.4. Effects of nickel chloride on electrophysiological effect of FeSO4 (200 µmol/L)

The TTCC channel blocker nickel chloride (NiCl2, 200 μmol/L)could not block the electrophysiological effects of FeSO4(200 μmol/L) (Table 4).

Table 1The electrophysiological effects of Fe2+ on the Slow reaction autorhythmic cells of the left ventricular outflow tract (Mean ± SD, n=8).

Table 2Effects of high concentration of Ca2+ on the electrophysiological effect of FeSO4 (200 μmol/L) (Mean ± SD, n=8).

Table 3Verapamil’s effects on electrophysiological effect of FeSO4 (200 μmol/L) (Mean ± SD, n=8).

Table 4Effects of nickel chloride on electrophysiological effects of FeSO4 (200 μmol/L) (Mean ± SD, n=8).

4. Discussion

Repeated blood transfusion, excessive consumption of high iron food and drugs can lead to the occurrence of iron overload, which can cause myocardial damage, arrhythmia and heart failure. Our previous studies showed that the electrophysiological characteristics of ventricular outflow tract resulting into slow response autorhythmic cells were one of the mechanisms of ventricular arrhythmias. In order to detect whether iron overload cause abnormal electrophysiological characteristics of slow response autorhythmic cells in ventricular outflow tract or not, we perfused the left ventricular outflow tract with different concentrations of FeSO4and detected changes in slow response action potential parameters of autoryhthmic cells. Results showed decreased Vmax, RPF ,VDD and APA, and increased APD50and APD90. MDP decreased too Indicating that iron overload could change the electrophysiological characteristics of slow reaction autorhythmic cells in left ventricular outflow tract. Our previous studies showed that phase 0 depolarization of ion current in slow response autorhythmic cell is mainly calcium influx together with a small amount of sodium ions influx. Repolarization process was the efflux of potassium ions. Phase 4 automatic depolarization is mainly L-type calcium current (ICa-L) participation and progressive attenuation of potassium efflux. And hyperpolarization-activated inward ion current(If) participates in pacemaker current[10,11]. The comparison and analysis of the above-mentioned results concluded that Fe2+may change the electrophysiological characteristics of the slow response cells of the left ventricular outflow tract by reducing the ICa. It was observed that electrophysiological effects of Fe2+could be partially inhibited by increasing the Ca2+concentration in the perfusion fluid, which further proved that Fe2+plays a role through the calcium channel. Perfusion of LTCC blockers, Verapamil, and Fe2+caused decrease of the electrophysiological effects of Fe2+indicating that Verapamil could inhibit the electrophysiological effects of Fe2+too. However, perfusion of TTCC blockers, nickel chloride could not decrease the electrophysiological characteristics of autorhythmic cells in the left ventricular outflow produced by Fe2+, indicating that nickel chloride could not inhibit the electrophysiological effects of Fe2+. The above-mentioned results showed that Fe2+may change the electrophysiological characteristics of the left ventricular outflow tract through LTCC.

Results of this study showed that when iron overload occurs Fe2+inflow, which goes directly through LTCC into autorhythmic cells in ventricular outflow tract, were in competition with the influx of calcium ions and then lead to shrinked L-type calcium current(ICa-L). This is probably the mechanism of electrophysiological characteristics of autorhythmic cells of left ventricular outflow tract produced by Fe2+. Under the above-mentioned condition, the left ventricle of the heart are prone to conduction abnormalities, which in turn causes tachycardia arrhythmia. This might be one of the mechanisms when ventricular arrhythmia occurs at iron overload.

Conflict of interest statement

The authors declare that there is no conflict of interest.

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