Alpha-Lipoic Acid Prevents Atrial Electrical and Structural Remodeling via Inhibition of NADPH Oxidase in a Rabbit Rapid Atrial Pacing Model
Lei Chen Xuzhou Medical College Afliated Hospital Wensu Chen Xuzhou Medical College Afliated Hospital Yao Zhang Xuzhou Medical College Afliated Hospital Zhirong Wang ( [email protected] ) Xuzhou Medical College Afliated Hospital https://orcid.org/0000-0001-9574-6240
Research article
Keywords: NADPH oxidase, Rapid atrial pacing, Reactive oxygen species, Oxidative stress, Alpha-lipoic acid
Posted Date: September 29th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-36814/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Page 1/22 Abstract
Background The pathogenesis of atrial fbrillation(AF) is complex, and the treatment method is still not satisfactory. A rapid atrial pacing (RAP) model was constructed to study the effects of alpha-lipoic acid (ALA) on electrical and structural remodeling, as well as its possible mechanism in rabbits.
Methods A total of 30 rabbits were randomly divided into a sham-operated group (SHAM group), a rapid atrial pacing model group (RAP group) and an alpha-lipoic acid+rapid atrial pacing model group (ALA+RAP group). Their right atriums were paced at a speed of 600 beats/min for 12 h in the RAP and ALA+RAP groups, and the atrial effective refractory period (AERP) and AERP frequency adaptability were determined during the pace. In each group, malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected to observe the effects of oxidative stress. The pathological structure of the atrial tissue was observed through HE and Masson staining. Ultrastructural changes in the atrial myocytes were observed by transmission electron microscopy (TEM), and the expression levels of Nox2 and Nox4 were detected by immunohistochemistry, western blot and ELISA.
Results Through testing AERP and AERP frequency adaptability, it was found that in the early stage of rapid atrial pacing, AERP gradually shortened, while ALA injection could remarkably delay this process. Correspondingly, AERP frequency adaptability in the RAP group was reduced, and ALA could enhance it. HE staining showed that pathological changes in the ALA+RAP group were milder than those in the RAP group. And it was found that in the ALA+RAP group, the deposition of collagen in the endomysium was remarkably reduced via Masson staining. Ultrastructure injury in the ALA+RAP group showed various degrees of improvement compared with the RAP group. RAP was accompanied by an increase in oxidative stress levels, and ALA could effectively inhibit RAP-induced oxidative stress in vivo via detecting SOD, MDA and ROS. In addition, Western blot showed that the expression of NOX2 and NOX4 was upregulated in RAP group, but ALA intervention could inhibit their expression. Moreover, immunohistochemistry and ELISA also got similar results as Western blot.
Conclusion ALA can inhibit atrial electrical remodeling and structural remodeling by reducing ROS production and alleviating oxidative stress injury induced by rapid right atrial pacing, and its mechanism may be related to inhibiting the activity of NADPH oxidase.
Background
Atrial fbrillation (AF) is one of the most common arrhythmias encountered in the clinic [1]. Atrial fbrillation-induced symptoms and complications can severely affect the quality of life and cause mortality [2-4]. Long-term maintenance of the sinus rhythm of AF remains a considerable challenge due to its complicated pathogenesis.
The available methods for controlling the atrial fbrillation rhythm include antiarrhythmic drugs and catheter ablation, both of which have obvious limitations [5, 6]. Upstream treatment of AF is a novel method introduced in recent years and it aims to achieve primary and secondary prevention of atrial
Page 2/22 fbrillation [7]. It was frst established in 2010 by the European Society of Cardiology to be a new method and strategy to prevent atrial fbrillation [8]. However, the precise mechanism of the genesis and development of AF remains incompletely known. Therefore, additional research on the genesis and maintenance of AF is of crucial importance.
A large number of studies have indicated that atrial remodeling is the central link for the genesis and maintenance of atrial fbrillation, which mainly includes atrial electrical remodeling, structural remodeling and contractile remodeling [9-11]. As research continues in recent years, growing evidence has indicated that oxidative stress plays an important role in atrial remodeling [12-14]. A large amount of reactive oxygen species (ROS) will be produced in the case of oxidative stress, which plays a vital role in AF [15- 17]. Consequently, effective regulation of ROS levels in vivo has become an important intervention target in the upstream treatment of AF.
ROS has numerous sources in vivo, but many studies have suggested that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is its primary source [18-20]. Plenty of evidence from clinical and experimental research has indicated that ROS plays a core role in numerous cardiovascular diseases (including AF). However, the clinical effects of antioxidant stress treatment are not satisfactory [21]. This may be because a general ROS scavenger can only neutralize one or several forms of ROS, while other ROS forms still exert a pro-arrhythmic effect. Therefore, a more comprehensive ROS scavenger or a targeted therapy specifc to each ROS source is required.
Alpha-lipoic acid (ALA) is a natural compound, one of the natural antioxidants with high activity [22]. Compared with other natural antioxidants such as vitamin C and carotene, ALA is water and lipid soluble, and it can exist in the cytoplasm and cell membrane. Moreover, it has been applied in diabetes [23], cardiovascular disease [24] and liver-kidney disease [25]. ALA can eliminate ROS and reduce the production of ROS by targeting NADPH oxidase [26-29]. Therefore, compared with general antioxidants, ALA can not only more comprehensively eliminate active substances in vivo but also target NADPH oxidase.
So far, research on the role of ALA in cardiovascular disease has been reported, but only a limited number of studies have reported on its effects on arrhythmia. This study constructed a RAP rabbit model to evaluate the effect of ALA on atrial remodeling in AF and its possible mechanism. Hopefully, this research can provide a new possibility for the clinical treatment of AF.
Methods
Chemicals
ALA was purchased from Yuanye Biotechnology (Shanghai, China). Rabbit polyclonal anti-NOX4 antibody was obtained from Novus Biologicals (Littleton, USA). Anti-NOX2 and goat anti-rabbit IgG/HRP were purchased from Bioss Biotechnology (Beijing, China). The reactive oxygen species (ROS) detection kits were purchased from Beyotime Biotechnology (Shanghai, China). The malondialdehyde (MDA)
Page 3/22 detection kits and superoxide dismutase (SOD) activity assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits for the protein Nox4 were purchased from Westang Biotechnology (Shanghai, China).
ALA solution preparation
ALA (80 mg/mL) was dissolved in 10% ethanol and sterilized by fltering. ALA solutions were prepared immediately before use.
Animals
In this study, 30 healthy New Zealand white rabbits weighing 2.9±0.2 kg (2.7–3.1 kg) were selected regardless of sex. All rabbits were randomly assigned to the SHAM group (n = 10), the RAP group (n = 10), or the ALA + RAP group (n = 10). All of them were fed adaptively in the Laboratory Animal Center for 7 d before experiments. In the ALA+RAP group, ALA (30 mg/kg) was administered intraperitoneally daily (i.p.) to the rabbits for 3 days before RAP [27]. In the remaining groups, equal volume of normal saline was injected intraperitoneally. All rabbits were obtained from Xuzhou Medical University Animal Center. This study was approved by the Animal Ethics Committee of Xuzhou Medical University Animal Center.
Construction of the RAP model
First, 1% pentobarbital sodium solvent was injected slowly through the ear vein at 2 ml/kg. After successful anesthesia induction, the anesthesia was maintained through an indwelling needle in the ear vein and a constant speed micropump. An incision was made in the middle of the neck, the trachea was isolated, tracheal intubation was conducted, and an animal ventilator was connected to assist in ventilation. The right internal jugular vein was isolated, and distal shaping of the 10-pole coronary sinus electrode was conducted, which was sent to the right atrium through the 6F sheathing canal, and the chest lead V1 was connected to the distal end of the coronary sinus buttcock line.
The BL-420s biological function experimental system was connected, and continuous stimuli were released at a frequency 10%–20% higher than the sinus rhythm. Microadjustment of the coronary sinus electrode direction was performed so that the stimulating electrode adhered to the right atrium. Then, 1:1 atrial and ventricular conduction observed simultaneously in the intracavity lead and the body surface lead electrocardiograms revealed complete atrial pacing. The position of the coronary sinus electrode was maintained, and 12 h continuous stimuli were carried out by pacing at a rate of 600 times/min at a pressure twice that of the pacing threshold.
After pacing, the rabbits were euthanized by intravenous injection of 100mg/kg pentobarbital sodium.
AERP determination
The 2-fold diastolic pacing threshold was treated as the output voltage, and the pulse width was set at 0.5 ms to measure AERP under the basic condition. Programmed premature extrastimulation (S1S2) was
Page 4/22 adopted at a stimulation frequency of 8:1. Decreasing scanning was carried out at S1S2 interphase at a step length of 10 ms and a scanning interval of 30 s. The refractory period was defned as the longest S1S2 interphase that S2 did not induce atrial activation in the time of S1S2 stimulation. AERP was measured 3 times, and the mean was treated as the baseline AERP value. AERP200 and AERP150 at the S1S1 of 200 ms and 150 ms were measured, respectively. The frequency adaptability is expressed as AERP200 − AERPl50.
Tissue processing [30]
The hearts were excised and washed immediately with buffered saline (pH 7.4) to remove any red blood cells and clots. The tissues were then weighed and homogenized in normal saline (0.9%) for 2 min. All homogenate samples were centrifuged for 20 min at 3,000 rpm at 4 °C in a refrigerated centrifuge to remove debris.
Detection of NOX4 and oxidative stress-related parameters
The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected using the respective assay kits. The levels of the NOX4 protein were determined by ELISA.
Histopathological analysis [30]
Samples of heart tissues were washed immediately with saline (0.9%) and fxed in 10% phosphate buffered formalin solution for 24 h. The tissues were then dehydrated in ascending grades of ethyl alcohol, cleared with xylene, and embedded in parafn. Parafn blocks were cut by a microtone to prepare 5 μm thick sections. Sections were stained with hematoxylin-eosin (H&E) to stain the nucleus and cytoplasm and with Masson’s trichome to stain collagen fbers for the detection of fbrosis.
Ultrastructural changes
After precooling the relevant instruments at 4°C, the samples of heart tissues were washed with saline (0.9%) then cut into 1×1×1 mm3 pieces and placed immediately in 4% glutaraldehyde at 4°C for 4 hours. Afterward, the samples were sent to the Neuroelectrometer Experimental Center of Xuzhou Medical University, sliced and stained, and observed by transmission electron microscopy.
Immunohistochemistry [30]
For the detection of p47 phox, parafn sections were deparafnized, rehydrated and washed in 0.05 M Tris-buffered saline (TBS) pH 7.6. Immunostaining was performed using a rabbit polyclonal antibody (p47-phox-H-195/sc-14015) at a dilution of 1:250 for 1 h at room temperature. Negative controls were performed by omission of the primary antibody. Then, the sections were incubated with NovoLink Polymer Detection System (product No. RE 7280-K; Leica Biosystems, Newcastle, UK) for 30 min to detect any tissue-bound primary antibody. Sections were further incubated with the substrate/chromogen, 3,3- diaminobenzidine (DAB), prepared from Novocastra DAB chromogen (50 μl) and 1 ml of NovoLink
Page 5/22 Substrate Buffer (Polymer). Washing with TBS (0.05 M, pH 7.6) twice for 5 min was performed following each step of incubation. Immunostained sections were then counterstained with Mayer’s hematoxylin. Finally, sections were dehydrated rapidly in ascending grades of ethanol, cleared in xylene, and mounted in DPX mounting medium. The stained sections were evaluated and photographed using an Olympus BX51 bright feld microscope equipped with an Olympus DP72 camera (Olympus Corporation, Japan).
Western blot
The whole-cell lysates of the cortical samples (N = 3 for each group) were obtained using tissue protein extraction kits supplemented with protease inhibitors (Roche, IN, USA). The total protein concentration was determined using BCA kits. An equal amount of protein was separated by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to nitrocellulose membranes. After washing with TBST, the blots were then incubated with the respective secondary antibodies for 2 h and developed by chemiluminescence. Relative optical densities of the blots were quantifed by densitometry.
Statistical analysis
SPSS 19.0 statistical software was adopted for statistical analysis. Measurement data were expressed as mean ± standard deviation. Data among multiple groups at all time points were compared using one- way analysis of variance. Differences in continuous repeated measuring data were compared by the repeated data analysis of variance. Further pairwise comparison was carried out using a Dunnett t-test. Differences were considered signifcant when P <0.05.
Results
1 Electrical remodeling-related indicators
1.1 Effect of ALA on AERP
Differences in AERP200ms and AERP150ms among the three groups under the basic condition (before pacing) were not signifcant (P>0.05). AERP200ms and AERP150ms had no obvious variation trend with an extension in time within 12 h in the SHAM group, while they were gradually shortened with the extension of pacing time within 12 h in both the RAP group and the ALA+RAP group (P<0.05). Compared with the SHAM group, AERP200ms and AERP150ms were signifcantly shortened after rapid pacing in the RAP group (P<0.05). Compared with the RAP group, AERP200ms and AERP150ms in the ALA+RAP group were increased at the corresponding time points (P<0.05). Thus, in the early stage of rapid atrial pacing, AERP gradually shortened, while ALA injection could remarkably delay this process (Tables 1-2).
Page 6/22 Table 1 Effect of alpha-lipoic acid on AERP200msin each group(n = 10, x±s, ms) Group 0 h 4 h 8 h 12 h
SHAM 115.60 ± 6.14 115.20 ± 6.09 115.95 ± 5.19 114.45 ± 7.58
RAP 116.84 ± 3.78 99.84 ± 5.05ab 87.92 ± 8.42ab 79.49 ± 7.83ab
ALA + RAP 116.92 ± 3.94 104.76 ± 9.96abc 100.77 ± 9.85abc 96.90 ± 4.43abc
a 푃< 0.05 compared with the 0 h of this group. b 푃< 0.05 compared with the SHAM group. c 푃< 0.05 compared with the RAP group.
Table 2 Effect of alpha-lipoic acid on AERP150ms in each group(n = 10, x±s, ms) Group 0 h 4 h 8 h 12 h
SHAM 104.24 ± 6.63 103.62 ± 6.06 104.63 ± 5.26 103.97 ± 6.41
RAP 105.83 ± 4.05 89.43 ± 5.00ab 80.29 ± 8.36ab 76.07 ± 7.88ab
ALA + RAP 105.57 ± 3.87 94.28 ± 10.13abc 91.87 ± 10.00abc 89.39 ± 4.78abc
a 푃< 0.05 compared with the 0 h of this group.b 푃< 0.05 compared with the SHAM group. c 푃< 0.05 compared with the RAP group.
1.2 Effect of ALA on AERP frequency adaptability
AERP frequency adaptability in the SHAM group indicated no distinct changes with an extension of pacing time within 12 h, while they were notably changed in the RAP group and the ALA+RAP group (P<0.05). Compared with the SHAM group, AERP frequency adaptability in the RAP group was shortened at corresponding time points (P<0.05). Compared with the RAP group, AERP frequency adaptability in the ALA+RAP group was increased at corresponding time points (P<0.05). AERP frequency adaptability was shortened in the ALA+RAP group at corresponding time points compared with the SHAM group (P<0.05). Thus, AERP frequency adaptability in the RAP group was reduced, and ALA could enhance it (Table 3).
Page 7/22 Table 3 Effect of alpha-lipoic acid on AERP frequency adaptability in each group(n = 10, x±s, ms) Group 0 h 4 h 8 h 12 h
SHAM 11.36 ± 0.90 11.58 ± 0.25 11.32 ± 0.28 11.49 ± 0.35
RAP 11.01 ± 2.29 10.41 ± 0.19b 7.62 ± 0.22ab 3.42 ± 0.36ab
ALA + RAP 11.35 ± 0.80 11.48 ± 0.30c 8.90 ± 0.25abc 7.26 ± 0.63abc
a 푃< 0.05 compared with the 0 h of this group.b 푃< 0.05 compared with the SHAM group. c 푃< 0.05 compared with the RAP group.
2 Structural remodeling-related indicators
2.1 HE staining
The rabbits in the SHAM group (Fig. 1a) showed orderly myocardial fbers and normal nuclei. Atrial muscle cell hypertrophy could be seen in the RAP group (Fig. 1b) along with irregularly sized nuclei, many nuclei showed pyknosis, there were disorderly myocardial fbers, and myocardial fbers rupturing and dissolving in some regions. Connective tissue accumulated between the muscle fbers, and there were gaps between myocardial cells. Pathological changes in the ALA+RAP group (Fig. 1c) were milder than those in the RAP group. Most of the nuclei appeared normal, the myocardial fbers did not show ruptures, and their arrangement was slightly ordered.
2.2 Masson staining
Figure 2 shows the Masson staining of the heart tissue. In the SHAM group (Fig. 2a), the heart had a small amount of collagen between the myocardium cells. Sections of hearts from the RAP group (Fig. 2b) showed a noticeable increase in collagen deposition in the connective tissue between the myocardial cells. In the ALA+RAP group (Fig. 2c), the deposition of collagen in the endomysium was remarkably reduced compared with the RAP group.
2.3 Effect of ALA on ultrastructural changes in each group
As shown in Figure 3, the tissue sections from the SHAM group (Fig. 3a) showed a normal ultrastructure, while it was severely injured in the RAP group (Fig. 3b), characterized by myoflament dissolution and mitochondrial swelling and deformation. Ultrastructure injury in the ALA+RAP group (Fig. 3c) showed various degrees of improvement compared with the RAP group (Fig. 3b).
2.3.1 Comparison of mitochondria: a small amount of mitochondrial aggregation could be seen in the rabbit atrial muscle cells in the SHAM group (Fig. 3a), which had even morphology and size and were regularly distributed. Mitochondria in the RAP group (Fig. 3b) were crowded, along with obvious swelling and deformity, an irregular distribution, and uneven morphology and size. The mitochondrial condition in Page 8/22 the ALA+RAP group (Fig. 3c) was improved compared with that in the RAP group (Fig. 3b), which displayed slightly regular distribution and relatively regular size and morphology.
2.3.2 Comparison of myofbrils: the myofbrils in the SHAM group (Fig. 3a) were orderly, and structural integrity and normal morphology of the sarcomere could be seen. Myofbrils in the RAP group (Fig. 3b) were disorderly; myoflament dissolution and incomplete sarcomere structures could be seen. The myofbril condition in the ALA+RAP group (Fig. 3c) was partly improved compared with that in the RAP group (Fig. 3b).
3 Effect of ALA on oxidative stress in each group
As shown in Table 4, compared with the SHAM group, the contents of MDA and ROS were signifcantly increased in the RAP group (P<0.05), while SOD activity was decreased (P<0.05). Compared with the RAP group, the contents of MDA and ROS were reduced in the ALA+RAP group (P<0.05), while SOD activity was enhanced (P<0.05). These results indicated that RAP was accompanied by an increase in oxidative stress levels, and ALA could effectively inhibit RAP-induced oxidative stress in vivo. Table 4 Effects of ALA on MDA、SOD and ROS in each group(n = 10, x±s) Group MDA(nmol/mgprot)༈mmol/mgprot༉ SOD(U/ mgprot) ROS(µmol/mg)
SHAM 1.30 ± 0.33 432.19 ± 2.65 0.004 ± 0.001
RAP 4.52 ± 0.28 a 107.50 ± 3.45 a 0.016 ± 0.002 a
ALA + RAP 2.44 ± 0.23ab 302.09 ± 6.14ab 0.009 ± 0.001ab
a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
4 Effects of ALA on NOX2 and NOX4 in each group
4.1 Western blot
Compared with the SHAM group, expression of NOX2 and NOX4 in the atrial muscle tissue in the RAP group was upregulated (P<0.05). Thus, RAP induced the upregulation of NOX2 and NOX4. Compared with the RAP group, expression of NOX2 and NOX4 in the atrial muscle tissue in the ALA+RAP group was decreased (P<0.05) compared with the SHAM group. Thus, it could be speculated that RAP induced an increase of NOX2 and NOX4, and ALA intervention could inhibit their expression (Tables 5-6, Fig. 4-5).
Page 9/22 Table 5 Effect of ALA on NOX2 Group NOX2/β-actin
SHAM 0.47 ± 0.09
RAP 1.12 ± 0.09a
ALA + RAP 0.68 ± 0.10ab
a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
Table 6 Effect of ALA on NOX4 Group NOX4/β-actin
SHAM 0.60 ± 0.12
RAP 1.14 ± 0.11a
ALA + RAP 0.82 ± 0.07ab
a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
4.2 Immunolocalization of NOX2
As shown in Fig. 6, the yellow staining indicates positive NOX2 expression. Compared with the SHAM group (Fig. 6a), the RAP group (Fig. 6b) had a markedly enlarged stained area, along with notably deepened yellow staining. Compared with the RAP group (Fig. 6b), the staining area in the ALA+RAP group (Fig. 6c) was distinctly reduced, and the yellow staining was notably lightened. Thus, RAP could enhance the NOX2 expression level, which could be reduced by ALA intervention.
5 ELISA
Compared with the SHAM group, the NADPH oxidase (NOX4) expression level in the RAP group was notably upregulated (P<0.05), and compared with the RAP group, the expression of NADPH oxidase (NOX4) in the ALA+RAP group was signifcantly downregulated (P<0.05). Compared with the SHAM group, the expression of NADPH oxidase (NOX4) in the ALA+RAP group was slightly upregulated (P<0.05). Thus, RAP could signifcantly increase NADPH oxidase (NOX4) expression, which could be improved in atrial tissue after ALA intervention (Table 7, Fig. 7).
Page 10/22 Table 7 Effect of ALA on NADPH oxidase (NOX4) Group NADPH oxidase(pg/ml,n = 10)
SHAM 1872.46 ± 161.73
RAP 3549.90 ± 413.94a
ALA + RAP 2348.67 ± 289.32ab
a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
Discussion
In this study, the frequency adaptability of AERP and AERP were measured to evaluate atrial electrical remodeling [31]. After RAP stimulation, electrical remodeling-related indicators in the RAP group developed remarkable changes in the early stage, which manifests as a shortened AERP and a loss of frequency adaptability. The HE and Masson staining, along with the SEM results, showed that compared with the SHAM group, rabbit atrial ultrastructure and pathological structures were notably changed in the RAP group. This further verifes that electrical remodeling and structural remodeling are closely related to the genesis and development of atrial fbrillation.
Oxidative stress participates in atrial electrical remodeling and structural remodeling, thus giving rise to lesions like atrial fbrosis [32-34]. This experiment constructed an RAP rabbit model, and atrial muscle oxidative stress-related indicators, namely, ROS, SOD and MDA, were detected to refect oxidative stress in the atrial muscle tissue at the time of atrial fbrillation. Among them, ROS is considered to be one of the major causes of oxidative stress [35], which plays a vital role in the genesis and maintenance of atrial fbrillation [36]. SOD is the most important enzyme in the frst-line cellular defense [37], and it is extensively distributed in all tissue and cells. MDA is the end byproduct of lipid peroxidation in oxidative stress [38], which is frequently treated as a detection index of lipid peroxidation degree. As was found in this experiment, among the oxidative stress-related indicators, the ROS and MDA content increased while SOD activity decreased in the RAP group compared with the control group. Thus, RAP can reduce SOD activity, destroying the frst-line defense of ROS and interfering with the balance between oxidation and antioxidation regulation, which then increases ROS and MDA content. These fndings indicated that RAP could induce oxidative stress injury, which has further verifed that oxidative stress is closely related to atrial fbrillation.
In addition, a large number of studies have reported that antioxidants can effectively improve atrial electrical remodeling and structural remodeling [39, 40]. However, their clinical effects remain unsatisfying. Therefore, we are looking forward to searching for a better upstream therapeutic agent or treatment for atrial fbrillation. NADPH oxidase is a major source of ROS in the body, the enhanced activity of which is closely associated with the genesis and development of atrial fbrillation [41-43]. In the NADPH oxidase family, NOX4 is an important subunit participating in the production of ROS [44, 45].
Page 11/22 Gp91phox (NOX2) and p22phox subunits are located on the plasma membrane, which can form active NADPH oxidase complexes when binding to several other subunits (including NOX4) in the cytoplasm, but NOX2 is its major functional subunit. In this experiment, compared with the SHAM group, the expression levels of nox2 and nox4 in the atrial muscle of the RAP group were distinctly upregulated. This suggests that RAP can enhance NADPH oxidase activity, thus promoting ROS production. Consequently, NADPH oxidase targeted therapy may be a novel effective treatment for preventing atrial fbrillation.
ALA is a naturally existing thiol antioxidant that can eliminate most of the active substances produced in the body as compared with general antioxidants. Meanwhile, it can inhibit NADPH oxidase. Research on the role of ALA in cardiovascular disease has increased gradually due to its unique features. It was discovered that ALA exerts antioxidative effects in cardiovascular diseases through multiple pathways, thus alleviating the genesis and progression of disease [46-49]. However, research on its role in arrhythmia, especially atrial fbrillation, is rarely reported. In this study, we observed oxidative stress- related indicators and discovered that the R+ALA group notably reduced the levels of oxidative stress induced by RAP. Through HE and Masson staining and the SEM results, compared with the RAP group, the cardiac fber structure in the R+ALA group was signifcantly improved. Comparisons of the AERP and AERP frequency adaptability among all groups showed that ALA could delay RAP-induced changes in AERP and AERP frequency adaptability. Furthermore, the results of immunohistochemistry, western blot and ELISA showed that compared with the RAP group, the R+ALA group had reduced expression of the NOX2 and NOX4 proteins. Consequently, we speculate that ALA can improve atrial electrical remodeling and structural remodeling in an ARP model, thus effectively preventing the genesis and progression of atrial fbrillation. Apart from antioxidative effects such as the direct inactivation of ROS, its mechanism of action may be to partially inhibit NADPH oxidase activity and reduce ROS production, thus improving atrial remodeling in this RAP model.
It was verifed in this experiment that ALA can directly inactivate ROS in an RAP rabbit model. In addition, it can partly inhibit NADPH oxidase activity and reduce ROS production, thus delaying the progression of atrial fbrillation. These results have provided a certain theoretical foundation for the antioxidant treatment of atrial fbrillation. However, this experiment is also associated with a specifc drawback, which is that the precise mechanism of action by which ALA inhibits NADPH oxidase activity, as well as the other antioxidation pathways, remains unclear.
Conclusions
This article demonstrates the electrical and structural remodeling of the atrium in a rapid atrial pacing model. This study used lipoic acid for the frst time in atrial rapid pacing models and proved that ALA intervention could inhibit atrial electrical remodeling and structural remodeling. Its mechanism may be to reduce oxidative stress injury by inhibiting the activity of NADPH oxidase.
Abbreviations
Page 12/22 ALA: Alpha-lipoic acid; AF: Atrial fbrillation; RAP: Rapid atrial pacing; AERP: Atrial effective refractory period; NADPH: nicotinamide adenine dinucleotide phosphate; HE: Hematoxylin-eosin; TEM: transmission electron microscopy; MDA: Malondialdehyde; ROS: Reactive oxygen specie; SOD: Superoxide dismutase
Declarations
Acknowledgments
Not applicable.
Consent for publication
Not applicable.
Funding
Not applicable.
Ethics approval and consent to participate
We have obtained written informed consent to use the animals in this study from the Animal Ethics Committee of Xuzhou Medical University (Xuzhou, china).
Availability of data and materials
The data sets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
LC, WC and YZ performed the experiments and analyzed the data. LC and ZW designed the study and wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Competing interests
The authors declare that they have no competing interests.
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Figures
Page 17/22 Figure 1
Light photomicrographs of the rabbit’s heart stained with hematoxylin and eosin（×400). (A) Myocardial fbers and nuclei in the SHAM group. (B) Myocardial fbers and nuclei in the RAP group. (C) Myocardial fbers and nuclei in the ALA+RAP group.
Figure 2
Photomicrographs of sections of the heart stained with Masson’s trichrome (×400). (A) The distribution of collagen in the SHAM group. (B) The deposition of collagen in the RAP group. (C) The deposition of collagen in the ALA+RAP group.
Page 18/22 Figure 3
Ultrastructural changes of the heart with transmission electron microscopy. (A) The ultrastructure in the SHAM group. (B) The ultrastructural changes in the RAP group. (C) The ultrastructural changes in the ALA+RAP group.
Page 19/22 Figure 4 a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
Figure 5 a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
Page 20/22 Figure 6
Immunolocalization of NOX2 in myocardial tissue (×400). (A) Immunolocalization of NOX2 in the SHAM group. (B) Immunolocalization of NOX2 in the RAP group. (C) Immunolocalization of NOX2 in the ALA+RAP group.
Page 21/22 Figure 7 a 푃< 0.05 compared with the SHAM group. b 푃< 0.05 compared with the RAP group.
Supplementary Files
This is a list of supplementary fles associated with this preprint. Click to download.
DATAAERP200ms.docx DATAAERP150ms.docx DATAAERPfrequencyadaptability.docx DATAMDASODROS.docx
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