Irisin Promotes Cardiac Homing of Intravenously Delivered MSCs and Protects against Ischemic Heart Injury

Abstract Few intravenously administered mesenchymal stromal cells (MSCs) engraft to the injured myocardium, thereby limiting their therapeutic efficacy for the treatment of ischemic heart injury. Here, it is found that irisin pretreatment increases the cardiac homing of adipose tissue‐derived MSCs (ADSCs) administered by single and multiple intravenous injections to mice with MI/R by more than fivefold, which subsequently increases their antiapoptotic, proangiogenic, and antifibrotic effects in rats and mice that underwent MI/R. RNA sequencing, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, and loss‐of‐function studies identified CSF2RB as a cytokine receptor that facilitates the chemotaxis of irisin‐treated ADSCs in the presence of CSF2, a chemokine that is significantly upregulated in the ischemic heart. Cardiac‐specific CSF2 knockdown blocked the cardiac homing and cardioprotection abilities of intravenously injected irisin‐treated ADSCs in mice subjected to MI/R. Moreover, irisin pretreatment reduced the apoptosis of hydrogen peroxide‐induced ADSCs and increased the paracrine proangiogenic effect of ADSCs. ERK1/2‐SOD2, and ERK1/2‐ANGPTL4 are responsible for the antiapoptotic and paracrine angiogenic effects of irisin‐treated ADSCs, respectively. Integrin αV/β5 is identified as the irisin receptor in ADSCs. These results provide compelling evidence that irisin pretreatment can be an effective means to optimize intravenously delivered MSCs as therapy for ischemic heart injury.

For the MI/R rat model, adult male and female Sprague-Dawley rats were randomly assigned to the following groups: Sham; MI/R+vehicle; MI/R+rADSCvehicle; and MI/R+rADSC-irisin.

ADSC preparation
ADSCs were isolated from adult male C57BL/6J mice and adult male Sprague-Dawley rats as we previously described [3] . Briefly, inguinal subcutaneous adipose tissue was removed under anesthesia. The adipose tissue was rinsed several times with phosphate buffered saline (PBS). Blood vessels were excised under a dissection microscope. The remaining adipose tissue was cut into fine pieces, digested with 0.1% collagenase type I at 37°C for 60 minutes, and centrifuged at 600 g for 10 minutes.
After red blood cell lysis with 1× lysis buffer, the cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 medium containing 10% fetal bovine serum (FBS) and penicillin-streptomycin. Six hours after cell plating, the medium was changed to remove nonadherent cells. Adherent cells were cultured in DMEM-F12-10% FBS and split several times for expansion. Cells from passages 2-3 were used in all experiments.

Adenovirus construction and transfection
The recombinant FNDC5 adenovirus was constructed by Likely Biotechnology (Beijing, China). Cells were infected with the FNDC5 adenovirus or adenovirus containing empty plasmids (control) for 24 hours at a multiplicity of infection (MOI) of 20-200. The medium was then replaced with fresh medium, and the cells were cultured for another 24 hours.

Adeno-associated virus serotype-9 construction and intramyocardial injection
Adeno-associated virus serotype-9 carrying CSF2 shRNA (AAV9-CSF2-shRNA) or scramble RNA (AAV9-control) was purchased from GenePharma. One month before the MI/R operation, AAV9-CSF2-shRNA or AAV9-control (2.6×10 10 vector genomes per mouse) was intramyocardially injected into the left ventricle free wall at three different sites. The experiments were performed by an investigator who was blinded to the group allocations.
Small interfering RNA (siRNA)-mediated gene knockdown ANGPTL4 siRNA, CSF2RB siRNA, SOD2 siRNA, and their scramble RNAs were purchased from GenePharma. When ADSCs reached 80% confluence, siRNAs were transfected into the cells with RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778075) according to the manufacturer's protocol (final siRNA concentration: 100 nM). After 8 hours of incubation (37°C), the transfection reagent-siRNA mixture was replaced with fresh growth medium. Successful knockdown was confirmed by Western blot analysis of the ANGPTL4, CSF2RB, and SOD2 protein levels.

ADSC labeling
Cultured ADSCs were labeled with the lipophilic dye CM-DiI (5 µM in DMEM-F12-10% FBS) for 20 minutes immediately before intravenous injection. ADSCs were also stained with cell plasma membrane staining kit with DiO, according to the manufacturer's protocol.

Flow cytometric analysis of the cardiac homing of ADSCs
The cardiac homing of ADSCs was evaluated by flow cytometry 1 day after the fifth intravenous injection (30 days after MI/R). Mice were heparinized (10 IU/g body weight), and 5 minutes later, they were anesthetized with an intraperitoneal injection of ketamine/xylazine mixture (100 mg/kg ketamine plus 10 mg/kg xylazine). Single cells were then enzymatically isolated from the hearts in 0.1% collagenase type II (30 minutes at 37°C) as we previously described [4] . A 'myocyte-depleted' cardiac cell population was prepared by filtering the single cells through a 40-μm mesh and was then stained with a PE/Cy5 anti-mouse/rat CD29 antibody (#102219, Biolegend). A PE/Cy5 Armenian hamster IgG isotype control antibody (#400909, Biolegend) was used as the negative control. The cardiac homing of ADSCs (CM-DiI+/CD29+) was determined with a flow cytometer (Epics MCL, Beckman).

The in vivo competitive homing assay
The competitive homing assay [5] was performed after a single intravenous injection (1 day after MI/R), and the distribution of ADSCs in organs was evaluated by flow cytometry. Vehicle-treated ADSCs (ADSC-vehicle) were labeled with DiO (a green dye) and irisin-treated ADSCs (ADSC-irisin) were labeled with CM-DiI (a red dye). Equal number of ADSC-vehicle-DiO and ADSC-irisin-DiI were then mixed before they were intravenously injected into the post-MI/R mice. An aliquot of the cell mixture was kept and analyzed by flow cytometry. The mice were sacrificed 1 day after intravenous injection (2 days after MI/R). The hearts, lungs, and spleens were removed from the mice. The hearts were prepared for the 'myocyte-depleted' cardiac cell population. The lungs were minced and then digested in 0.1% collagenase type II (20 minutes at 37°C). Spleens were homogenized into a single-cell suspension using PBS.
After red blood cell lysis with 1× lysis buffer, the lung cells and splenocytes were resuspended in PBS. Flow cytometry assays were performed to detect ADSC-vehicle-DiO and ADSC-irisin-DiI in the 'myocyte-depleted' cardiac cell population and singlecell suspensions of lung and spleen.

Echocardiography
M-mode images of mice and rats subjected to 1-2% isoflurane anesthesia were obtained via a VisualSonics 770 echocardiography machine (Canada) as previously described [6] . Hearts were viewed along the long and short axes between the two papillary muscles. The LV end-systolic diameter (LVESD) and LV end-diastolic dimension (LVEDD) were measured. The LVEF was automatically calculated by echocardiography software as follows: Measurements were performed for at least five separate cardiac cycles per mouse.
Echocardiography was performed by a single experienced operator in a blinded fashion.

Evaluation of angiogenesis
For the evaluation of angiogenesis, heart sections were deparaffinized and subjected to antigen retrieval in hot citric acid buffer. After cooling, the slides were permeabilized with 0.2% Triton-100 for 15 minutes, blocked with 1% BSA in PBS for 2 hours, and incubated with an anti-CD31 primary antibody at 4°C overnight (#GB13063, Servicebio). A donkey anti-goat antibody conjugated with CY3 (#GB21404, Servicebio) served as the secondary antibody. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, GB1012, Servicebio). Images of immunostained sections were acquired with a Nikon Eclipse C1 microscope and Nikon DS-U3 camera. The myocardial capillary density was quantified by Image-Pro Plus 6.0 software (Media Cybernetics). The representative image for each group was selected based upon the mean value.

Determination of apoptosis
ADSC apoptosis was determined by TUNEL staining with a One Step TUNEL Apoptosis Assay Kit (Beyotime, C1090). Images were acquired with a Nikon Eclipse Ni microscope and Nikon DS-Ri2 camera. Apoptosis of cardiomyocytes in heart tissue was determined with a Roche In Situ Cell Death Detection Kit (Sigma, 11767305001 and 11767291910) according to the manufacturer's protocol. Images were acquired with a Nikon Eclipse C1 microscope and Nikon DS-U3 camera. The index of apoptosis was determined by the number of TUNEL-positive nuclei/total nuclei. The representative image for each group was selected based on the mean value.

Masson's trichrome staining
Hearts were harvested from anesthetized mice and rats and embedded in paraffin.
The heart tissue extending from just distal to the coronary ligation point to the apex was separated into different segments at 200 µm intervals. Serial 5-µm-thick sections were obtained from each segment for Masson's trichrome staining according to the manufacturer's protocol (Sigma, HT15). Microscopic images of mouse heart sections were obtained with a 1.25× object lens (Nikon, Japan). Images of rat heart sections were acquired with a Nikon Eclipse C1 microscope and Nikon DS-U3 camera. For quantification, measurements of 5 transverse heart sections were analyzed. Fibrotic size was determined as the average ratio of the fibrotic area to the LV area (fibrotic size %) with Image-Pro Plus 6.0 software (Media Cybernetics).

CM collection
We employed a modified method to prepare the CM from ADSCs [3] . CM was generated as follows: after growth to 90% confluence in 6-well dishes, ADSCs were pretreated with 100 ng/mL irisin or vehicle for 1 day. The culture medium was washed and replaced with serum-free DMEM/F12 medium. Twenty-four hours later, the serumfree DMEM/F12 medium was collected and centrifuged at 1000 g for 10 minutes to obtain the supernatant (CM).

In vitro cardiomyocyte apoptosis assay
Both primary NRVCs and iPSC-CMs were used for the in vitro cardiomyocyte apoptosis assay. Primary cultures of NRVCs from 1-to 2-day-old Sprague-Dawley pups were prepared as described previously [7] . Human iPSC-CMs were purchased from HELP Therapeutics (#NC20010435). The differentiation and preparation of iPSC-CMs have been described previously [8] . For the evaluation of cardiomyocyte apoptosis, cultured NRVCs and iPSC-CM were subjected to 6 hours of H2O2 (200 µM) before treatment with recombinant irisin (100 ng/mL), ANGPTL4 (2 μg/mL), or CM derived from ADSCs.

Capillary-like tube formation assay
rCAECs were purchased from Procell (CP-R081, Wuhan, China) and cultured in low-glucose DMEM containing endothelial cell growth supplement (#211-GS, Cell Applications), 10% FBS, and penicillin-streptomycin. For the tube formation assay, Matrigel (BD Biosciences) was added to each well of a 48-well plate and allowed to polymerize at 37°C for 30 minutes. rCAECs were treated with CM derived from ADSCs for 24 hours. Images of the formation of capillary-like structures were obtained by computer-assisted microscopy. The total length per field was calculated from five random fields.

Immunohistochemistry
For fixed tissues, wax blocks were cut into 5-μm-thick sections and mounted on glass slides for staining. The slides were deparaffinized and subjected to antigen retrieval in hot citric acid buffer. After cooling, the slides were permeabilized with 0.2% Triton-100 for 15 minutes, blocked with 1% BSA in PBS for 2 hours, and incubated overnight at 4°C with an anti-troponin T mouse monoclonal antibody (Thermo Fisher Scientific, MS-295-P0) (1/1,000). The primary antibody was visualized with a donkey anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Fluor 488 (A77440, Yeasen, China). The frozen sections were fixed in acetone at 4°C for 15 min. The sections were blocked with PBS containing 1% BSA at room temperature. Sections were then incubated overnight at 4°C with an anti-TNNT2 rabbit polyclonal antibody (Affinity Biosciences, DF6261). The primary antibody was visualized with a goat anti-rabbit IgG (H+L) secondary antibody conjugated with Alexa Fluor 488 (33106ES60, Yeasen, China). Nuclei were stained with DAPI (GB1012, Servicebio). Images were acquired with a Nikon Eclipse C1 microscope and Nikon DS-U3 camera.

RNA sequencing analysis
Differential gene expression analysis was performed using RNAseq at Shanghai Biotree Biological Technology [9] . After treatment with vehicle or irisin for 24 hours, total RNA was extracted from ADSCs via an RNeasy Mini Kit (Qiagen, 74106) according to the manufacturer's protocol. RNA purity was assessed using a NanoPhotometer® spectrophotometer (Implen). RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies).
A total of 1 µg of RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer's recommendations, and index codes were added to attribute sequences to each sample.
Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature conditions in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, an NEBNext adaptor with hairpin loop structures was ligated to prepare for hybridization. For the preferential selection of cDNA fragments 250~300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 µL of USER Enzyme (NEB, USA) was incubated with size-selected, adaptor-ligated cDNA for 15 minutes at 37°C and then at 95°C for 5 min prior to performing PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. The library preparations were sequenced on an Illumina NovaSeq platform, and 150 bp paired-end reads were generated. Differential expression analysis was performed using the DESeq2 R package (1.16.1). DESeq2 provides statistical routines for determining differential expression among digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, during which gene length bias was corrected. GO terms with corrected P values less than 0.05 were considered to be significantly enriched by the differentially expressed genes. The KEGG database is a resource for understanding the high-level functions and utilities of biological systems, such as cells, organisms and ecosystems, based on molecular-level information, especially from large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). We used the clusterProfiler R package to assess the statistical enrichment of differentially expressed genes in KEGG pathways. The thermal cycling conditions were as follows: denaturation at 95°C for 5 minutes, followed by 40 cycles of 10 s at 95°C, 20 s at 55°C, and 20 s at 72°C.

Western blot analysis
Total proteins were isolated from cells or heart tissues with 1× lysis buffer (CST #9803) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, 78438). A total of 30-70 μg of protein per sample was separated via gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blocked with 5% milk for 1 hour. The membrane was incubated overnight with primary antibodies at 4°C. After incubation with a secondary HRP-conjugated anti-mouse antibody (Abbkine, A21010, 1/10,000) or anti-rabbit antibody (Abbkine, A21020, 1/10,000) at room temperature for 2 hours, the membranes were exposed to enhanced chemiluminescent (ECL) substrate            Tables   Table I: Genes of the cytokine-cytokine receptor interaction pathway with