Exosomal miR-320d derived from adipose tissue-derived MSCs inhibits apoptosis in cardiomyocytes with atrial fibrillation (AF)

Abstract MicroRNAs (miRNAs) play a key role in various pathological processes like atrial fibrillation (AF), which is a common cardiac arrhythmia. Exosomes are essential information carrier in the intercellular communication. Therefore, this study aimed to investigate the effects of exosomal miR-320d on cardiomyocytes with AF and related mechanisms. To do this, AMSCs were transfected with miR-320d mimics, AMSCs-derived exosomes were co-cultured with cardiomyocytes with AF. MTT, TUNEL staining, flow cytometry, real-time PCR, western blots, and luciferase reporter assays were performed. The results revealed that miR-320d expression was decreased in AF cardiomyocytes. AF increased apoptosis and reduced cell viability in cardiomyocytes. By transfection with miR-320d mimics, the miR-320d level was increased in AMSCs, exosomes and cardiomyocytes, which reversed the effect of AF on cardiomyocytes. STAT3 was down-regulated in AF cardiomyocytes and was a direct target gene of miR-320d. Inhibition of STAT3 abolished the effect of modified exosomes in cardiomyocytes, causing decreased apoptosis and increased cell viability. Taken together, the results suggested that exosomal miR-320d was associated with AF cardiomyocytes apoptosis and cell viability and that the effect of miR-320d on cardiomyocytes is STAT3-dependent. Therefore, this study provides a novel understanding of the molecular basis of AF and provides insight into therapeutic strategies for AF.


Introduction
Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting about 1% of young patients and up to 9% of patients at least 75 years old [1]. In patients with or without cardiovascular conditions, AF is induced by triggers in the pulmonary vein [2]. Reports suggest that AF is associated with increased cardiovascular morbidity and mortality [3]. From the aetiological point of view, AF is a manifestation of complex aberrancies in multiple levels, including molecular, cellular, electrical and structural alterations [4]. Furthermore, various risk factors may cause AF, such as heart failure, hypertension, and mitral valve disease [5].
Growing evidence reveals that AF is related to structural and electrical remodelling that are both critical for the initiation and maintenance of AF [6,7]. Moreover, apoptosis, a type of programmed cell death, is a complex process of cell self-destruction, characterizing abnormal cell shape, membrane blebbing and chromatin degradation [8]. In AF studies, atrial HL-1 cardiomyocytes with tachypacing showed enhanced caspase activity which suggests increased apoptosis [9]. In congestive heart failure-induced rapid atrial pacing models, atrial cells were observed to have a high level of apoptosis [10,11], implying the close correlation between the high ventricular rate and cell death in AF.
Exosomes are specialized endocytic-origin vesicles in nano-size [12] and play essential roles in intercellular communication via DNA, RNAs and proteins [13]. To date, exosomes have been widely applied as the carrier to deliver functional microRNAs (miRNAs) to the recipient cells in vitro [14,15]. Adipose tissue-derived mesenchymal stem cells (AMSCs) are regarded as an ideal source to generate exosomes [16]. Thus, AMSCs can be a promising mediator to deliver miRNAs in exosome-mediated intercellular communication [16]. For example, miR-181-5P transfected-AMSCs engineered exosomes to inhibit hepatic fibrosis in hepatic stellate cells [17]. Also, exosomes generated from miR-122-treated AMSCs sensitize the hepatocellular carcinoma cells when given a chemotherapy treatment [18]. Due to its high efficacy, it may be worthwhile to apply AMSCs-exosome approach to AF patients.
In addition to the relationship between miRNAs and exosomes, miRNAs, 21-24 nucleotides, are a class of endogenous and small non-coding RNAs that exert essential roles in the regulation of gene expression [19]. As an important posttranscriptional regulator, miRNAs can bind 3 0 -UTR of target genes, thereby either repressing subsequent translation or inducing mRNA degradation [20]. It is becoming more evident that miRNAs play significant roles in the regulatory mechanisms to modulate various biological processes [19].
In AF patients, miR-26 significantly reduces AF, leading to increased K þ current which is a hallmark of atrial electrical remodelling [21]. The muscle-specific miRNA, miR-133, has been reported to regulate cardiac ion channels via downregulating the ether-a-go-go-related gene protein (ERG) level and K þ current [22]. Also, miR-320d, a member of the miR-320 family, is associated with cell growth and apoptosis, for example, in glioma cells, overexpression of miR-320d inhibits cell growth while enhances apoptosis [23]. Furthermore, forced expression of miR-320d increases staurosporine and tumour necrosis factor a-induced apoptosis in smooth muscle cells [24]. However, the effect of miR-320d on cardiomyocytes is still unclear. Therefore, the objectives of this study were to investigate the function of miR-320d in cardiomyocytes of AF mice and whether AMSCs-exosome is an effective way to deliver miR-320d to cardiomyocytes.

Animal
In this study, experimental protocols were approved by the Ethical Review Board of Cangzhou Central Hospital. All animal procedures and care were performed according to the Animal Care and Treatment Administration of the National Ministry of Health and the Ethics Committee in Cangzhou Central Hospital. Healthy male C57BL/6 mice (24.2 ± 2.1 g, 8 weeks) were purchased from the Animal Center Laboratory at Peking University (Beijing, China). All mice were individually housed at 20-26 C with 40-70% relative humidity and a 12:12-h light-dark cycle (6:00 am to 6:00 pm). Water and food were given ad libitum.

AF induction
Wild-type mice were anesthetized via intraperitoneal injection of sodium pentobarbital (dose: 40 mg/kg) and supported by artificial respiration. Body temperature was kept at 37 C and monitored for the entire procedure. The AF induction protocol has been previously described [25]. The criteria of successful AF induction were defined as rapid irregular atrial rhythm lasting more than one second.

Cardiomyocytes isolation
Primary neonatal mice myocytes were isolated from newborn heart ventricles by trypsin solution. Briefly, hearts of newborn C57BL/6 mice were isolated aseptically after decapitation. The hearts were then cultured in serum-free Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan) and were digested by 0.25% trypsin solution. The cell suspension was centrifuged at 2500 rpm for 3 min and resuspended in DMEM containing 10% foetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA). The non-adherent cardiomyocytes were plated into non-coated culture flasks (Thermo Fisher Scientific, Waltham, MA, USA). Cardiomyocytes were cultured under a condition of 5% CO 2 at 37 C.

AMSC exosomes isolation
After 48-h transfection, AMSCs-derived exosomes were isolated from the AMSCs supernatant using the ExoQuick-TC Kit (System Biosciences, Mountain View, CA, USA) in accordance with the manufacturer's instructions. The AMSCs-derived exosomes were assessed by western blots of exosome surface markers: CD9, CD63 and CD105 (BD Biosciences Pharmingen, San Diego, CA, USA) [29]. Precipitates from AMSCs DMEM were used as the negative control. Exosome pellets were resuspended in PBS solution at a 5 lg/lL protein concentration.

Cardiomyocytes transfection
The miR-320d mimics and negative control were obtained from RiboBio (Guangzhou, China). The signal transducer and activator of transcription-3 (STAT3)-containing pCDNA3.1 and STAT3-siRNA (25 nmol/L) were obtained from Invitrogen (Invitrogen, Shanghai, China). The transfection procedure was performed using the Lipofectamine TM 2000 Transfection Reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. The transfection efficacy was evaluated by real-time PCR or western blots.

TUNEL staining
Cardiomyocytes were collected and fixed in 4% paraformaldehyde in PBS with 0.12 mM sucrose for 20 min. The apoptosis level was assessed by a TUNEL apoptosis detection kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Apoptosis was visualized via fluorescence microscopy (AmScope, Irvine, CA, USA).

Flow cytometry
Cardiomyocyte apoptosis was measured by using an Annexin Vnexin Vn was measured bas mea (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturers' instructions. Annexin V-FITC/propidium iodide (PI) and FITC positive cardiomyocytes were evaluated by a flow cytometry assay (BD Biosciences, San Diego, CA, USA).

Luciferase reporter assay
The target gene of miR-320d was predicted by the online tool, TargetScan (www.targetscan.org). For this study, STAT3 was predicted to be a potential target gene of miR-320d. The luciferase reporter assay (Promega, Fitchburg, WI, USA) was performed to identify if STAT3 3 0 -UTR sequence contained the miR-320d binding site. The protocol was performed in accordance with the manufacturers' instructions.

Western blot
Total proteins were isolated using cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; 8-12%) and then were transferred to polyvinylidene difluoride (PVDF) membranes. Next, PVDF membranes were blocked with 5% skimmed milk in PBS (0.05%) Tween 20 for 2 h at room temperature. The primary antibodies of STAT3, caspase-3, Bax, Bcl-2 and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Quantification was assessed by the Uvitec Alliance software (Eppendorf, Hamburg, Germany).

Statistical analysis
Data were presented as mean ± standard error. Statistical analysis was performed using SPSS v.18.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed with Student's t-test. Difference was considered to be significant at p < .05. The sample size in each treatment group was 3 for the entire study.

Identification of AMSCs and AMSCs-derived exosomes
In this study, AMSCs were isolated from subcutaneous adipose tissue. In general, CD molecules were used as cell surface markers to identify the AMSCs. The results revealed that CD44, CD73, CD90, CD105 and CD166 were up-regulated while CD34 and CD45 were down-regulated in AMSCs ( Figure  1(A)), suggesting that the AMSCs were correctly isolated from mice adipose tissues. Then, AMSCs were given adipogenic induction treatment for 14 days. According to the images from Oil Red O positive staining, AMSCs could differentiate to adipose tissue (Figure 1(B)), which suggests AMSCs have normal differentiation potential. Furthermore, surface markers of exosomes were detected and the results revealed that the expressions of CD9, CD63 and CD81 significantly increased in AMSCs-derived exosomes (Figure 1(C)), suggesting these exosomes could be used for subsequent experiments.

Down-regulation of miR-320d promoted apoptosis in cardiomyocytes with AF
Annexin V-FITC/PI and TUNEL staining assays revealed that cardiomyocytes with AF displayed elevated levels of apoptosis (Figure 2(A,B)). Using the MTT assay, reduced cell viability was found in cardiomyocytes (Figure 2(C)). Meanwhile, miR-321d expression was decreased in cardiomyocytes with AF compared to control cells (Figure 2(D)).

Apoptosis reduced in cardiomyocytes with AF cocultured with modified exosomes
When cardiomyocytes with AF was administrated to miR-320d mimics-modified exosomes, the expression of miR-320d was increased (Figure 3(A)), leading to dampened apoptosis (Figure 3(B,C)) and increased cell viability (Figure 3(D)). These results suggest that the effect of AF on cardiomyocytes was reversed from increased expression of miR-320d induced by engineered exosomes. To further explore the mechanisms underlying miR-320d-related apoptosis, western blots were performed to detect apoptosis-associated proteins. The results revealed that expression of Bax and caspase-3 increased while the level of Bcl-2 reduced in cardiomyocytes with AF (Figure 3(E)), suggesting that the Bax/Bcl-2/caspase-3 pathway may mediate AF-induced apoptosis in cardiomyocytes.

MiR-320d promoted apoptosis in cardiomyocytes by targeting STAT3
In cardiomyocytes, the expression of STAT3 was increased ( Figure 4(A,B)). Through the online analysis tool, TargetScan (www.targetscan.org), a binding site of miR-320d was found in the 3 0 -UTR of STAT3 (Figure 4(C)). The luciferase reporter assay was performed to determine the mechanic relationship between STAT3 and miR-320d. The results indicated that the luciferase activity was decreased in cardiomyocytes with the wild-type 3 0 -UTR sequence while there was no change of luciferase activity in cells with the mutant 3 0 -UTR sequence (Figure 4(D)), which suggested STAT3 was a target gene of miR-320d in cardiomyocytes with AF.

Down-regulation of STAT3 inhibited apoptosis in cardiomyocytes with AF
To further investigate the function of STAT3 in cardiomyocytes with AF, STAT3-siRNA was applied to cardiomyocytes treated with miR-320d mimics-modified exosomes. The expression of STAT3 was significantly decreased (Figure 5(A)). In addition, the apoptosis level ( Figure 5(B,C)) was increased while cell viability ( Figure 5(D)) was decreased in STAT3-siRNA-treated cardiomyocytes. These results together suggest that STAT3 functionally participates in the miR-320dassociated cellular activities in cardiomyocytes with AF.

Discussion
As the most common cardiac arrhythmia, AF affects a wide range of populations worldwide [1]. The occurrence of AF is becoming increasingly prevalent with higher incidence as age increases, leading to a greater number of medical and social costs, including high mortality, frequent hospitalizations, low life quality and substantial health care expenditures [2,31]. Therefore, there is an urgent need to explore the mechanisms underlying AF and develop new therapies to prevent and treat AF. In this study, miR-320d was decreased in cardiomyocytes with AF, causing elevated apoptosis and suppressed cell viability. Delivery of miR-320 mimics via engineered AMSCs-derived exosomes increased miR-320d expression and reversed its effect on cardiomyocytes. Also, STAT3 was a direct target of miR-320d, mediating the functions of miR-320d in cardiomyocytes with AF.
MiR-320d is a relatively less-studied member of the miR-320 family. Early studies report that the aberrant expression of miR-320d was found in various cancer cell lines and tissues, such as acute myeloid leukemia [32], breast cancer [33] and colorectal cancer [34]. In the present study, the increased expression of miR-320d was detected in cardiomyocytes with AF and was associated with elevated apoptosis and inhibited cell viability. This is consistent with previous studies regarding the role of miR-320d in apoptosis. Qin et al. [23] reported that miR-320d is a biomarker for prognosis in glioma tissues and cells and the up-regulation of miR-320d is associated with enhanced apoptosis in glioma tissues and cell lines via inhibiting the matrix metalloproteinase family. Moreover, norcantharidin induces miR-320d-dependent apoptosis in prostate cancer cells via targeting leukemia-1 (Mcl-1) [35]. Collectively, miR-320d may play an important regulatory role in apoptosis in various cell types and may be a promising target to modulate apoptosis.
One of the primary goals in this study was to determine whether AMSCs-exosome was an effective delivery system to transport miR-320d to cardiomyocytes, thereby affecting cardiomyocyte activities. One important way of intercellular communication is the delivery of functional molecules via exosomes which has been widely applied to modulate cellular activities, thereby regulating disease progression and rehabilitation [14]. For example, MSCs-originated exosomes enriched with miRNA-let7c attenuates renal fibrosis [36]. Also, exosomes with forced expression of miR-146 leads to increased survival rate and M2 polarization in mouse sepsis [37]. In the present study, miR-320d mimics were transfected into AMSCs and exosomes, which then significantly decreased miR-320d expression in cardiomyocytes with AF. Notably, cardiomyocytes co-cultured with miR-320d mimicsmodified exosomes expressed opposite cell activities, including reduced apoptosis and increased viability. These results suggest that AMSCs-derived exosomes were an efficient way to deliver functional molecules to impact the cardiomyocyte activity, providing a potential therapeutic approach for AF.
Apoptosis-associated cardiomyocyte death has been extensively studied in various cardiovascular diseases, including postnatal cardiogenesis, idiopathic dilated cardiomyopathy, arrhythmogenic ventricular dysplasia and heart failure [38]; however, the effects of apoptosis on AF needs to be further elucidated. In this study, the downstream signalling underlying the roles of miR-320d in cardiomyocyte apoptosis with AF were further explored. The results revealed that STAT3 was the direct target of miR-320d and the inhibition of STAT3 could reverse the effect of miR-320d in cardiomyocytes with AF. Moreover, STAT3 is activated in a contextdependent pattern in various biological processes, such as proliferation, development, differentiation and apoptosis [39]. With regard to AF, STAT3 is up-regulated which mediates the structural and electrical remodelling in the rapid pacinginduced AF model [40]. Activation of STAT3 signalling is also reported to contribute to the inflammation process of AF [41]. Taken all together, the enhanced STAT3 expression may result from the down-regulation of miR-320d in AF, suggesting a potential signalling pathway to suppress the effect of STAT3 in AF.
In conclusion, the results in the present study demonstrated that down-regulated miR-320d promoted cardiomyocyte apoptosis and inhibited cell viability through targeting STAT3. Delivery of miR-320d mimics via AMSC-exosome reversed the effects of miR-320d on cardiomyocytes with AF. Therefore, this study may provide new insights into the mechanism underlying AF and provide a promising approach to modulate cardiomyocyte activity via exosome-mediated intercellular communication.

Disclosure statement
No potential conflict of interest was reported by the authors.