Mitophagy is an autophagic response and plays essential roles in survival, development, and homeostasis of cells. It has beenreported that mitophagic dysfunction is involved in several cardiovascular diseases. However, the effect of mitophagy on atrialfibrillation (AF) is still unknown. .erefore, we investigated the exact role of mitophagy in human chronic AF. Western blot wasused to detect the protein abundance. .e mitochondrial morphology and structure were observed by transmission electronmicroscopy. Immunofluorescent stainings were performed to analyze colocalization of mitochondria with autophagosomes orlysosomes. Totally, 43 patients with valvular heart disease undergoing cardiac surgery were selected, including 21 patients withchronic AF. Comparing with the sinus rhythm (SR) group, we found the size and number of mitochondria in atrial myocytes ofpatients with AF increased significantly. In addition, expression of LC3B II and LC3B II/LC3B I ratio was significantly decreased inthe AF group. Moreover, the expression of p62 was markedly elevated in the AF group compared with that in the SR group. .eresults of immunofluorescence staining and western blot showed an enhanced expression of Cox IV in the AF group. Dualimmunofluorescent stainings revealed that mitophagy defect in atrial myocytes of patients with AF resulted from dysfunction inthe process of delivery of mitochondria into autophagosomes. For the first time, impaired mitophagy, during the phagocytosis ofmitochondria, is associated with human chronic AF. Mitophagy could be a potential therapeutic target for AF.
1. Introduction
Atrial fibrillation (AF) is the most common arrhythmia,which increases the risk of stroke and heart failure and isassociated with significantly increased mortality [1, 2].However, the pathogenesis of AF is still not fully understood,and the intervention is challenging. Mitochondria are keyplayers in cardiomyocyte energy metabolism, redox stateregulation and apoptosis. Recently, increasing evidencereveals that mitochondrial dysfunction provides anarrhythmogenic substrate for initiation and perpetuation ofAF, such as oxidative stress, energy metabolic dysregulation,and calcium homeostasis imbalance [3–5].
Autophagy is a conserved cellular pathway that ischaracterized by the engulfment of cargo into double-
membrane autophagosomes, which then fuse with lyso-somes for degradation. Autophagy plays essential roles insurvival, development, and homeostasis of cells. To ensuresurvival, the cell uses autophagy to selectively digest proteinaggregates, oxidized lipids, and damaged organelles [6, 7].Mitophagy is an autophagic response that specifically targetsdamaged, and hence potentially cytotoxic, mitochondria,which plays a crucial role in the mitochondrial qualitycontrol and maintenance of mitochondrial function [8]. Ithas been reported that mitophagic dysfunction is involved inseveral cardiovascular diseases in the animal model, such asatherosclerosis, cardiac hypertrophy, and cardiomyopathy[8]. Recently, some studies have indicated that autophagydysfunction contributes to AF [9–12]. However, the effect ofmitophagy on AF is still unknown. .erefore, in this study,
HindawiCardiology Research and PracticeVolume 2020, Article ID 6757350, 9 pageshttps://doi.org/10.1155/2020/6757350
we investigated the exact role of mitophagy in humanchronic AF.
2. Materials and Methods
2.1. Collection of Human Atrial Samples. A total of 43 pa-tients with valvular heart disease undergoing cardiac surgerywere selected and divided into a chronic AF group (21 caseswith long-standing persistent AF) and a sinus rhythm (SR)group (22 cases; Table 1). .e diagnosis of AF was achievedby evaluating medical records and 12-lead electrocardio-gram findings. .ose who had hypertension, diabetesmellitus, coronary heart disease, infective endocarditis, ac-tive rheumatism, pulmonary disease, hyperthyroidism, orautoimmune disease were excluded from this study. .eprotocol was in accordance with the Helsinki Declarationand was approved by the Human Ethics Committee of theFirst Affiliated Hospital of Guangxi Medical University(Guangxi, China). All patients enrolled in this study pro-vided written informed consent.
Right atrial appendage (RAA) tissues weighing about100mg were removed at the beginning of the surgical in-terventions under extracorporeal circulation. A portion ofthe tissues was fixed in 2.5% glutaraldehyde for ultra-structure analysis under transmission electron microscopy..e remainder of the tissues were snap-frozen in liquidnitrogen for protein isolation and frozen sections to performan immunofluorescence staining.
2.2.Western Blot. Total proteins were extracted from tissuesby RIPA buffer (Beyotime Institute of Biotechnology,Shanghai, China) with PMSF (Sigma-Aldrich, St. Louis, Mo.,USA). Protein concentration was determined with a BCAprotein assay kit (Beyotime Institute of Biotechnology,Shanghai, China). .e proteins were boiled with 5X SDS-PAGE sample loading buffer (Beyotime Institute of Bio-technology, Shanghai, China) and separated by 8%, 10%, or12% SDS-PAGE, followed by transfer onto a 0.22 μm pol-yvinylidene difluoride membrane (EMDMillipore, Billerica,MA, USA) using the Mini Trans-Blot electrophoretictransfer cell system (Bio-Rad Laboratories, Inc., Hercules,CA, USA). .e membranes were blocked for 1 h at roomtemperature with 5% nonfat milk in TBST (20mMTris-HCl,0.5MNaCl, 0.1% Tween 20) and incubated with anti-COX IIantibody (Abcam, Cambridge, UK) diluted at 1 : 5,000, anti-COX IV antibody (Cell Signaling Technology, Danvers, MA,USA) diluted at 1 :1,000, anti-LC3B antibody (Abcam,Cambridge, UK) diluted at 1 : 2,000, or anti-p62 antibody(Cell Signaling Technology, Danvers, MA, USA) diluted at1 :1,000 overnight at 4°C. Subsequently, the membraneswere incubated with IRDye 700CW goat anti-rabbit or anti-mouse IgG (LI-COR Biotechnology, Lincoln, NE, USA)diluted at 1 :10,000 for 1 hour at room temperature. .esignals were visualized and quantified with the Odysseysystem (LI-COR Biosciences, St. Charles, USA). Proteinband intensities were expressed relative to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), which was incubatedwith anti-GAPDH antibody (Abcam, Cambridge, UK) di-luted at 1 :10,000 overnight at 4°C.
2.3. Immunofluorescence Staining. .e frozen RAA tissueswere embedded in the optimum cutting temperature (OCT)compound and sectioned at −25°C in a cryostat. Sections(5 μm in thickness) were fixed with 4% polyformaldehyde for15 minutes at room temperature. .e sections were per-meabilized with 0.1% TritonX-100 for 20 minutes followedby blocking with 10% goat serum for 1 hour at roomtemperature. .en, the sections were incubated with anti-Cox IV antibody (Cell Signaling Technology, Danvers, MA,USA) diluted at 1 : 200, anti-LAMP-1 antibody (Cell Sig-naling Technology, Danvers, MA, USA) diluted at 1 : 200, oranti-LC3B antibody (Abcam) diluted at 1 :100 overnight at4°C. Subsequently, the sections were incubated with AlexaFluor 488-conjugated goat anti-mouse antibody (Cell Sig-naling Technology, Danvers, MA, USA) diluted at 1 : 500, orAlexa Fluor 594-conjugated goat anti-rabbit antibody (CellSignaling Technology, Danvers, MA, USA) diluted at 1 : 500for 1 hour at room temperature. DAPI (Solarbio Science &Technology, Beijing, China) was used to stain the cell nuclei.Finally, the fluorescence images were captured by using alaser scanning microscope and further analyzed byImagePro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD,USA).
2.4. Mitochondrial Morphology Analysis. .e mitochondrialmorphology and structure were observed by transmissionelectron microscopy. .e RAA tissues were cut into1× 1× 1mm sized pieces and fixed with 2.5% glutaraldehydefor 4 hours. .en, the samples were washed with 0.1M
Table 1: .e characteristics of patients with SR or AF.
Characteristics SR group(n� 22)
AF group(n� 21) p values
Age (years) 50.32± 10.14 53.29± 7.63 0.286Gender (male) 12 (54.54) 9 (42.86) 0.443Cardiac disease type
Note. Values expressed as mean± SD or n (%). SR, sinus rhythm; AF, atrialfibrillation; MS, mitral stenosis; MI, mitral inadequacy; AI, aortic inade-quacy; CVL, combined valvular lesion; LAD, left atrial diameter; LVEF, leftventricular ejection fraction; NYHA, New York Heart Association.
2 Cardiology Research and Practice
phosphate-buffered saline (PBS) and postfixed by 1% os-mium tetroxide solution for 2 hours. Subsequently, thesamples were dehydrated in an ethanol gradient series (100,90, 80, 75, and 50%) and 100% acetone, immersed in pro-pylene oxide, and embedded in epoxy resin at 60°C. Ul-trathin sections were obtained by using a Leica EM UC6rtultramicrotome (Leica Microsystems, Inc., Wetzlar, Ger-many) and were doubly stained with 0.1% uranyl acetate and3% lead citrate solution. .e sections were observed andphotographed by using a transmission electron microscope(H-7650, Hitachi, Ltd., Tokyo, Japan). A further statisticalanalysis was performed by Image J _v1.8.0 (Media Cyber-netics, Inc., Bethesda, MD, USA).
2.5. Detection of Colocalization of Mitochondria with Auto-phagosomes or Lysosomes. Dual immunofluorescent stain-ings were performed to analyze colocalization ofmitochondria with autophagosomes or lysosomes. As weknow, Cox IV is an inner mitochondrial membrane protein.We used Cox IV to identify mitochondria. Cox IV waslabeled with Alexa Fluor 488-conjugated goat anti-mouseantibody showing green fluorescence, which representedmitochondrial mass and localization. LAMP-1 that is anintegral glycoprotein residing in lysosomes was used foridentification of lysosomes [13]. LAMP-1 was labeled withAlexa Fluor 594-conjugated goat anti-rabbit antibodyshowing red fluorescence, which represented lysosomalmass and localization. LC3 was labeled with Alexa Fluor 594-conjugated goat anti-rabbit antibody showing red fluores-cence and used for identification of autophagosomes. .emerged image of Cox IV and LC3 that showed yellowfluorescence indicated the engulfment of mitochondria intoautophagosomes..emerged image of Cox IV and LAMP-1that showed yellow fluorescence indicated the infusion ofautophagosomes enclosing mitochondria with lysosomes..e fluorescence images were captured by using a laserscanning microscope, and the fluorescence intensity wasquantified by ImagePro Plus 6.0 (Media Cybernetics, Inc.,Bethesda, MD, USA).
2.6. Statistical Analysis. .e continuous data are presentedas mean± standard deviation (SD). .e discrete data areshown as percentages. .e continuous variables between 2groups were analyzed by the unpaired Student’s t-test. .ediscrete variables between 2 groups were analyzed by theChi-square test. p< 0.05 was considered to indicate astatistically significant difference. All statistical analyseswere performed using SPSS 16.0 (SPSS Inc., Chicago, IL,USA).
3. Results
3.1. Impaired Cardiac Autophagy in Patients with ChronicAtrial Fibrillation. To explore the role of autophagy on AF,the autophagy markers LC3B and p62 were measured in theSR group and the AF group, respectively. .e results ofimmunofluorescence staining suggested a decreased ex-pression of LC3B in the AF group (p � 0.046; Figure 1(a)).
Consistently, the results of western blot revealed that ex-pression of LC3BII and the LC3BII/LC3BI ratio were bothsignificantly decreased in the AF group compared with thatin the SR group (p � 0.013 and p � 0.03, respectively;Figure 1(b)). Moreover, the expression of p62 was markedlyelevated in the AF group compared with that in the SR group(p � 0.014; Figure 1(b)). .ese results indicated that therewas a cardiac autophagy defect in the patients with chronicAF.
3.2. Mitochondria Accumulation and Increased AutophagicVacuoles in Atrial Myocytes of Patients with Chronic AF..e differences of mitochondrial morphology and numberbetween the SR group and the AF group were observed bytransmission electron microscopy. We found that the mi-tochondria were larger, and their numbers were obviouslyincreased in atrial myocytes of patients in the AF group,compared with those in the SR group (p � 0.001 andp � 0.001, respectively; Figure 2(a)). In addition, there was asignificant increase in autophagic vacuoles and decrease ininfusion autophagosomes with lysosomes (p � 0.023 andp � 0.047, respectively; Figure 2(b)). .ese results demon-strated that the mitophagy in atrial myocytes of patients withchronic AF may be impaired.
3.3. Defective Mitophagy in Atrial Myocytes of Patients withChronicAF. Mitophagy is an autophagic response and playsa crucial role in the mitochondrial quality control process.To observe the change in mitophagy in patients with chronicAF, Cox II and Cox IV, which are localized to the innermitochondrial membrane and are used as marks formitophagy [14, 15], were determined in the SR group and theAF group, respectively. .e results of immunofluorescencestaining showed an enhanced expression of Cox IV in the AFgroup (p � 0.024; Figure 3(a)). Consistently, the results ofwestern blot showed that the expression of Cox II and CoxIV was significantly increased in the AF group, comparedwith that in the SR group (p � 0.001 and p � 0.032, re-spectively; Figure 3(b)). .ese findings further verified thatthere was a defective mitophagy in atrial myocytes of pa-tients with chronic AF.
3.4. Decreased Engulfment of Mitochondria into Autophago-somes in Atrial Myocytes of Patients with Chronic AF. Toinvestigate the underlying mechanism of mitophagy defect inchronic AF patients, the mitophagy flux was detected by dualimmunofluorescent stainings. .e results showed that theyellow fluorescence intensity caused by merge of Cox IV andLC3 was obviously decreased in the AF group compared withthat in the SR group (p � 0.046; Figure 4(a)). However, theyellow fluorescence intensity caused by merge of Cox IV andLAMP-1 had no significant difference between the SR groupand the AF group (p � 0.485; Figure 4(b)). .ese findingssuggested that mitophagy defects in atrial myocytes of pa-tients with chronic AF may result from dysfunction in theprocess of delivery of mitochondria into autophagosomes.
Cardiology Research and Practice 3
4. Discussion
In the present study, we found impaired mitophagy inpatients with chronic AF and further demonstrated thatdysfunction in the process of engulfment of mitochondriainto autophagic vesicles results in defective mitophagy. Tothe best of our knowledge, this is the first study to report thatimpaired mitophagy is associated with human chronic AF.Mitophagy could be a potential therapeutic target for AF.
In this study, a decreased LC3BII level and LC3BII/LC3BI ratio were observed in RAA tissues of patients withchronic AF. Meanwhile, an elevated p62 level was found inchronic AF patients. .ese findings indicated impaired
cardiac autophagy in chronic AF patients. Moreover, a re-duction in LC3B expression in chronic AF patients, revealedby immunofluorescence staining, implied that the formationof autophagosomes was deceased. .is finding furtherverified that there is impaired cardiac autophagy in chronicAF patients. Dual immunofluorescent staining confirmedthe decreased engulfment of mitochondria into autopha-gosomes in atrial myocytes of patients with chronic AF.
Intact autophagic responses are crucial to preservingcardiovascular homeostasis in physiological conditions [8].However, the role of autophagy in AF is still controversial.Yuan et al. [10] found activation of AMPK-dependentautophagy in AF patients and the rapid atrial pacing canine
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Figure 1: Impaired cardiac autophagy in patients with chronic AF. (a) Representative immunofluorescence images and semiquantitativeanalysis of LC3B in RAA (n� 9 per group). (b) Representative western blots and quantification of protein expressions of LC3B and p62 inRAA (the SR group, n� 16; the AF group, n� 18). Scale bar� 20 μm. ∗p< 0.05 vs the SR group. SR, sinus rhythm; AF, atrial fibrillation; RAA,right atrial appendage.
4 Cardiology Research and Practice
model. Recently, they reported that activation of autophagycontributed to AF by promoting degradation of L-typecalcium channels [12]. Wiersma et al. [9] found thatautophagy was activated upon endoplasmic reticulum (ER)stress and contributed to cardiomyocyte remodeling inexperimental and human AF. Inhibition of ER stress wasshown to protect against cardiac remodeling in in vitro andin vivo models of AF by preventing autophagy activation.However, a recent study including the largest clinical
samples so far showed that impaired cardiac autophagy isassociated with developing postoperative AF in patients aftercoronary artery bypass surgery [11]. In addition, anotherstudy revealed that ablation of NLRP3 inflammasome im-proved autophagy and reduced cardiac damage with pro-tection of the prolongation of the age-dependent PR interval,which is associated with AF by cardiovascular aging [16].How to interpret the contradictory results in these studies?We think that it is necessary to recognize the dynamic nature
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Figure 2: Mitochondria accumulation and increased autophagic vacuoles in atrial myocytes of patients with chronic AF. (a) Representativeelectron microscopic images of mitochondria in RAA (n� 5 per group, scale bar� 1 μm)..e asterisk indicates typical mitochondrial. (b, d)Semiquantitative analysis of mitochondrial area and quantity of mitochondria in RAA. (c) Representative electron microscopic images ofautophagosomes and autophagic vacuoles in RAA (n� 5 per group, scale bar� 200 nm). .e autophagosome containing mitochondria ismarked with a single arrow. .e autophagic vacuole is marked with double arrows. (e, f ) Semiquantitative analysis of autophagosomes andautophagic vacuoles in RAA. ∗p< 0.05, ∗∗p< 0.01 vs the SR group. SR, sinus rhythm; AF, atrial fibrillation; RAA, right atrial appendage.
Cardiology Research and Practice 5
of autophagy. Shirakabe et al. [17] reported that autophagywas upregulated between 1 and 12 hours after transverseaortic constriction (TAC) in mice but was downregulatedbelow physiological levels 5 days after TAC. .is resultsuggests that autophagy is dynamic in the process of dis-eases. Activation of autophagy may be an adaptive responseto pressure overload in the early stage, but autophagy in-activation after its temporary activation promotes the de-velopment of cardiac hypertrophy and heart failure [18]..epatients of the AF group enrolled in this study had long-
standing persistent AF. .e degree of atrial fibrosis is moresevere in long-standing persistent AF [19]. Atrial fibrosisappears as a common endpoint in a variety of AF-promotingconditions, including senescence, heart failure, mitral val-vular disease, and myocardial ischemia [20]. Recently, someevidence indicated that impaired cardiac autophagy wasassociated with cardiac fibrosis. .erefore, we speculate thatautophagy is defective in the late stage of AF, and impairedautophagy may contribute to AF by prompting atrial fi-brosis. .e activated autophagy observed in AF may be an
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Figure 3: Defective mitophagy in atrial myocytes of patients with chronic AF. (a) Representative immunofluorescence images andsemiquantitative analysis of Cox IV in RAA (n� 9 per group, scale bar� 20 μm). (b) Representative western blots and quantification ofprotein expressions of Cox II and Cox IV in RAA (the SR group, n� 14; the AF group, n� 18). ∗p< 0.05, ∗∗p< 0.01 vs the SR group. SR, sinusrhythm; AF, atrial fibrillation; RAA, right atrial appendage.
6 Cardiology Research and Practice
adaptive response to stress for maintaining cardiac ho-meostasis. However, it is harmful to cardiomyocytes whenthis response is excessive. .e potential mechanisms onautophagy from adaptive to maladaptive response need to beinvestigated in the future. .erefore, we suggest that the roleof autophagy depending on the stage of AF should be ex-plored, and to which extent autophagy activation is bene-ficial for preventing AF should also be elucidated.
On the other hand, the autophagy process includes threeparts: autophagy vesicle formation, engulfment of cargo, andfusion with lysosomes. At the beginning of autophagy, anautophagy vesicle with a double membrane is formed toencapsulate protein molecules or/and organelles that need tobe degraded. .e autophagic vesicles form autophagicbodies after wrapping the material to be degraded. Finally,lysosomes and autophagic bodies fuse into autophagic ly-sosomes, which use the internal hydrolases to degrade theencapsulated cell components [21]. Impaired function of theabove three parts may cause autophagy instability and celldysfunction. Mitochondrial homeostasis is an important
mechanism for maintaining mitochondrial function in cells.Even in normal cells, mitochondria are also constantlyfusing and dividing to maintain mitochondrial homeostasis.
Mitophagy is a special autophagy and plays a crucial rolein the mitochondrial quality control and maintenance ofmitochondrial function [8]. Early studies have confirmedthat mitophagy can adjust the number of mitochondria inthe cell to match the metabolic needs and, at the same time,remove the damaged mitochondria to complete the qualitycontrol of mitochondria [22, 23]. Hence, mitophagy isconsidered to be essential for maintaining cardiomyocytehomeostasis and viability in the basal state [24]. In thepresent study, increasedmitochondria size and number werefound in atrial myocytes of patients in chronic AF bytransmission electron microscopy. .ese results indicateddysfunction in mitochondrial metabolism and qualitycontrol. Meanwhile, significantly increased autophagicvacuoles and decreased infusion autophagosomes with ly-sosomes were observed, which suggested the mitochondrialdysfunction may be associated with impaired mitophagy in
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Figure 4: Decreased engulfment of mitochondria into autophagosomes in atrial myocytes of patients with chronic AF (n� 4 per group, scalebar� 20 μm). (a) Representative dual immunofluorescence images and semiquantitative analysis colocalization of Cox IV and LC3B in RAA.(b) Representative dual immunofluorescence images and semiquantitative analysis colocalization of Cox IV and LAMP-1 in RAA. ∗p< 0.05vs the SR group. SR, sinus rhythm; AF, atrial fibrillation; RAA, right atrial appendage.
Cardiology Research and Practice 7
atrial myocytes of patients in chronic AF. Increased ex-pression of COX II and COX IV in the AF group furtherrevealed that there was a defective mitophagy in atrialmyocytes of patients with chronic AF. Consistent with ourfindings, Wiersma et al. [4] found myolysis and dispersedmitochondria in patients with persistent AF and reducedcellular ATP levels in patients with long-standing persistentAF. As a result, increased damaged mitochondria contrib-uted to the generation of reactive oxygen species (ROS) viadamage-associated molecular patterns (DAMPs) or theCAMK II pathway, which in turn regulated the cell functionand led to enhanced activation of the NLRP3 inflammasome[25, 26]. Active NLRP3 inflammasomes could induce therelease of inflammatory factors and production of AF-re-lated extracellular matrix (such as collagen). In addition,ROS and oxidative stress are considered as a novel mech-anism of AF [27]. Hence, elevated damaged mitochondriacould be a key upstream target in the development of AF.
However, why did the mitophagy defect happen? .edual immunofluorescent stainings were used to investigatethe underlying mechanism of mitophagy defect in chronicAF patients. Cox IV, LC3, and LAMP-1 were used foridentification of mitochondria, autophagosomes, and lyso-somes, respectively. .e results showed that the fluorescenceintensity caused by merge of Cox IV and LC3 was obviouslydecreased in the AF group, but the fluorescence intensitycaused by merge of Cox IV and LAMP-1 was not signifi-cantly different between the SR group and the AF group..ese findings suggested that mitophagy defects in atrialmyocytes of patients with chronic AF may be due to dys-function in the process of delivery of mitochondria intoautophagosomes. However, our findings still need more in-depth studies to testify.
Collectively, our finding provided convincing experi-mental evidence, for the first time, that impaired mitophagy,during the phagocytosis of mitochondrial, is associated withhuman chronic AF. Mitophagy could be not only a potentialtherapeutic target for AF but also a promising mechanism ofAF, which is suitable for further exploration.
Data Availability
.e data used to support the findings of this study weresupplied by Zhiyuan Jiang under license. Requests for accessto these data should be made to Zhiyuan Jiang (e-mail:[email protected]).
Conflicts of Interest
.e authors declare that they have no conflicts of interest.
Authors’ Contributions
Shuang Zhou and Zhiyuan Jiang performed the experi-ments. Weiran Dai and Shuang Zhou wrote the manuscript.Guoqiang Zhong helped in the design of the experimentsand critically revised the manuscript. Shuang Zhou andWeiran Dai contributed equally to this work. GuoqiangZhong and Zhiyuan Jiang are co-correspondents.
Acknowledgments
.e authors would like to thank their teacher Dr. Zhong andeveryone in their team for their hard work. .is work wassupported by the Natural Science Foundation of China(NSFC) (Nos. 81760060 and 81660054) and Guangxi NaturalScience Foundation (No. 2017GXNSFAA198053)..is workwas also supported by the First Affiliated Hospital ofGuangxi Medical University.
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