Haemin pre‐treatment augments the cardiac protection of mesenchymal stem cells by inhibiting mitochondrial fission and improving survival

Abstract The cardiac protection of mesenchymal stem cell (MSC) transplantation for myocardial infarction (MI) is largely hampered by low cell survival. Haem oxygenase 1 (HO‐1) plays a critical role in regulation of cell survival under many stress conditions. This study aimed to investigate whether pre‐treatment with haemin, a potent HO‐1 inducer, would promote the survival of MSCs under serum deprivation and hypoxia (SD/H) and enhance the cardioprotective effects of MSCs in MI. Bone marrow (BM)‐MSCs were pretreated with or without haemin and then exposed to SD/H. The mitochondrial morphology of MSCs was determined by MitoTracker staining. BM‐MSCs and haemin‐pretreated BM‐MSCs were transplanted into the peri‐infarct region in MI mice. SD/H induced mitochondrial fragmentation, as shown by increased mitochondrial fission and apoptosis of BM‐MSCs. Pre‐treatment with haemin greatly inhibited SD/H‐induced mitochondrial fragmentation and apoptosis of BM‐MSCs. These effects were partially abrogated by knocking down HO‐1. At 4 weeks after transplantation, compared with BM‐MSCs, haemin‐pretreated BM‐MSCs had greatly improved the heart function of mice with MI. These cardioprotective effects were associated with increased cell survival, decreased cardiomyocytes apoptosis and enhanced angiogenesis. Collectively, our study identifies haemin as a regulator of MSC survival and suggests a novel strategy for improving MSC‐based therapy for MI.


| INTRODUC TI ON
Despite the advanced developments in surgical treatment and pharmacological therapy, myocardial infarction (MI) is still a major cause of morbidity and mortality worldwide. 1 Mesenchymal stem cell (MSC)-based therapy has shown promising results in MI treatment because of the capacity of MSCs to differentiate into cardiomyocytes and confer paracrine effects. The efficacy of MSC-based therapy is nonetheless seriously restricted by poor cell survival in the hostile environment of the injured heart. [2][3][4] Oxidative stress in the ischaemic heart can quickly induce apoptosis of transplanted MSCs. 2 It has been reported that fewer than 1% of MSCs can survive in the ischaemic rat heart after MI at 24 hours after transplantation. 5 Therefore, exploring a novel strategy to enhance the retention and engraftment of MSCs in the ischaemic heart is urgently needed. Indeed, several pre-treatment strategies, including hypoxia and genetic modification, have shown to increase the survival of MSCs under hostile environment. 6,7 Cell death is mainly mediated by mitochondrial function, which is closely related to mitochondrial dynamics. 8 Mitochondria undergo fusion and fission to form a network for maintaining cell function. 9,10 Mitochondrial fusion is regulated by mitofusin 1 (Mfn1) and Mfn2, whereas mitochondrial fission is mainly regulated by mitochondrial fission protein dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (Fis1). Converging evidence has shown that mitochondrial fission results in fragmented mitochondria and thus induces apoptosis. 11,12 Nevertheless, whether ischaemic conditions can induce mitochondrial fission and thus lead to apoptosis of transplanted MSCs has not been determined.
Haem oxygenase 1 (HO-1), an inducible stress protein, possesses cytoprotective defences including antioxidative stress, antiapoptosis and anti-inflammation functions during challenge by different stressors. 13,14 A previous study has shown that HO-1 up-regulation inhibits mitochondrial fission, thus attenuating apoptosis of cardiomyocytes induced by intermittent hypoxia. 15 Furthermore, cardiac-specific overexpression of HO-1 significantly reduces up-regulated mitochondrial fission and therefore protects against doxorubicin-induced dilated cardiomyopathy. 16 Given that HO-1 plays a critical role in regulating mitochondrial dynamics, we have been suggested that the ischaemic condition induces apoptosis of MSCs via up-regulation of mitochondrial fission which is regulated by HO-1. Therefore, pre-treatment with haemin, an HO-1 inducer, can increase the capability of MSCs to tolerate ischaemic conditions via inhibition of mitochondrial fission and thus enhance cardioprotective effects that ameliorate the damage from MI.

| Cell culture
Human bone marrow (BM)-MSCs were purchased from Cambrex BioScience (catalog no. PT-2501). BM-MSCs were routinely cultured as previously described. 17 Cells were passaged at a ratio of 1:3 when they reached confluence. The cells from passages 3-4 were used in the current study.

| Serum deprivation and hypoxia (SD/H)exposed cell culture and haemin pre-treatment
To mimic the ischaemic conditions in vitro, BM-MSCs were cultured under SD/H challenge. 18 In brief, when BM-MSCs reached 70%-80% confluence, the completed culture medium was changed to medium without foetal bovine serum (FBS) and then cultured under hypoxia (1% oxygen, 5% carbon dioxide and 94% nitrogen) for 48 hours. For haemin pre-treatment, BM-MSCs were cultured in complete medium with 10 µM haemin under normoxia (95% air and 5% carbon dioxide) for 24 hours prior to SD/H challenge.

| siRNA transfection
Control siRNA or HO-1 siRNA was used to transfect BM-MSCs using Lipofectamine RNAiMAX (13778-075; Invitrogen). Briefly, control siRNA or HO-1 siRNA was diluted with OptiMEM and mixed with the transfection reagent. Each mixture was added to BM-MSCs at 70%-80% confluence and then incubated for 24-48 hours. Finally, the transfection efficiency was examined by Western blot analysis.

| MitoTracker staining
The morphology of mitochondria was examined by MitoTracker staining as previously reported. 15

| TUNEL staining
Apoptosis of BM-MSC after different treatments was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining kit (11684795910; Roche). Briefly, after different treatments, the cells were washed with PBS, fixed and incubated with 1 µg/mL of Proteinase K/10 mmol/L Tris solution for 15 minutes at room temperature. Following washing with PBS twice, the cells were incubated with the TUNEL reaction mixture for 1 hour at 37°C in a dark place. Finally, the cells were washed and mounted with DAPI to stain the nuclei. Images of five different view fields for each slide were randomly captured (magnification of 20x). The apoptosis of BM-MSCs was calculated as the proportion of positive TUNEL cells to total DAPI-positive cells.

| Western blot analysis
The protein of each sample was extracted using RIPA buffer (9806, CST), and then the amount of concentrated protein was measured.

| Preparation of conditioned medium and HUVEC tube formation analysis
The conditioned medium (CdM) of MSCs was collected as previously described. 19 Briefly, BM-MSCs with or without haemin pretreatment were seeded in 6-well plated and cultured until 70%-80% confluence. Subsequently, the medium was replaced with 2 mL per well serum-free medium. After 48 hours culture, the CdM was collected, centrifuged and stored at −80°C until use. HUVECs (30 000 cells/well) were seeded in a 96-well plate coated with growth-factor-reduced matrigel (BD Biosciences, 356230). Next, HUVECs were treated with CdM derived from BM-MSCs and haemin-BM-MSCs.
After 6 hours of treatment, capillary-like tube formation was imaged (magnification of 10x). The endothelial tube length and branching points were analysed using ImageJ software. The experiments were repeated at least three times.

| Echocardiography assessment
The heart function of each mouse from the different groups was evaluated by transthoracic echocardiography (Ultramark 9; Soma TechnologyA) at 4 weeks after cell transplantation. The echocardiographic parameters were analysed using MATLAB R2011b software (MathWorks).

| Masson's trichrome staining
After echocardiography evaluation, all mice were killed, and the hearts were collected. The mouse hearts were fixed, embedded and sectioned into 5 μm sections. Fibrosis in the mouse hearts was detected by Masson's Trichrome Stain Kit (HT15; Sigma). Images of each slide were captured (magnification of 4x). The percentage of the infarct size was analysed as follows: (fibrosis area/total left ventricle area)×100%.

| Immunohistochemistry
Immunohistochemical staining was performed as previously described. 3 Briefly, the heart sections were hydrated, the antigen was retrieved, and the specimen was blocked with 5% bovine serum albumin for 30 minutes. Subsequently, heart sections were stained with the following primary antibodies, anti-HNA (ab191181, Abcam) and anti-CD31 (77 699, CST), at a 1:100 dilution and then incubated overnight at 4°C. After washing, the slides were incubated for 30 minutes with streptavidin peroxidase-conjugated secondary antibody (ab64264, Abcam) at room temperature. After this incubation, the slides were washed three times in PBS, and the antibody complexes were coloured with diaminobenzidine and then counterstained with haematoxylin. Five sections were randomly collected from each mouse, and six mice from each group were captured (magnification of 10x).

| Polymerase chain reaction
Human Alu-sx repeat sequences in the heart tissue from the different groups were evaluated by genomic polymerase chain reaction (PCR) as previously described. 3 The primer of human Alu-sx was F:5'-GGCGCGGTGGCTCACG-3', R:5'-TTTTTTGAGACGGAGTCTCGCTC-3.
The product was detected by electrophoresis in 1.5% agarose gel supplemented with ethidium bromide.

| Statistical analysis
Values are shown as the mean ± SEM. Statistical analyses were performed using Prism 5.04 software (GraphPad Software Inc.).
The comparison between two groups was analysed using unpaired Student's t tests and between multiple groups using one-way ANOVA followed by the Bonferroni test. A P value <0.05 was considered statistically significant.

| Haemin suppresses SD/H-induced mitochondrial fission and apoptosis of BM-MSCs
To test the protective effects of haemin on BM-MSCs, we pretreated BM-MSCs with different concentration of haemin (1, 5, 10, 20 μmol/L) for 24 hours and then exposed them to SD/H. The CCK-8 assay showed that haemin pre-treatment greatly enhanced the viability of BM-MSCs under SD/H in a dose-dependent manner and 10 μmol/L haemin pre-treatment exhibited the best protective effects ( Figure 1A). Furthermore, we pretreated BM-MSCs with 10 μmol/L haemin with different time (6, 12, 24, 48 hours) and then exposed them to SD/H. The CCK-8 assay also showed that

| Haemin inhibits mitochondrial fragmentation and apoptosis of BM-MSCs by regulating HO-1
As haemin is an HO-1 inducer, we investigated whether the protec-

| Haemin-pretreated BM-MSCs improved cell survival in mouse hearts following MI
We first performed anti-HNA staining to detect cell survival at 4 weeks after transplantation. Both BM-MSCs and haemin-pretreated BM-MSCs were detected in ischaemic heart tissue, with a

| Haemin-pretreated BM-MSCs inhibited the apoptosis of cardiomyocytes and improved angiogenesis in mouse hearts following MI
The apoptosis of cardiomyocytes among the different groups was assessed by TUNEL staining. Compared with the sham group, the apoptosis of cardiomyocytes was dramatically increased in the MI group ( Figure 5A,B). MSC transplantation greatly inhibited the apoptosis of cardiomyocytes, and haemin-BM-MSCs were superior to BM-MSCs in attenuating the apoptosis of cardiomyocytes in the ischaemic hearts of mice ( Figure 5A,B). The capillary density of the ischaemic area among the different groups was detected by CD31 staining. The capillary density was decreased in the MI group compared with the sham group ( Figure 5C,D). The capillary density of the ischaemic area increased following MSC treatment ( Figure 5C,D).
Notably, the haemin-BM-MSC group had a much higher capillary density than the BM-MSC group ( Figure 5C

| D ISCUSS I ON
This study presents several major findings ( Figure 6). MI is a major contributor to the mobility and mortality of people with cardiovascular diseases, accounting for 11.2% of deaths worldwide. 21 The ischaemic condition caused by insufficient blood flow leads to a marked loss of cardiomyocytes in the heart. Furthermore, Mitochondria dynamics play an essential role in inducing cell death. 30 Mitochondrial fusion leads to elongated mitochondria, whereas mitochondrial fission produces small round mitochondria. 10 There is a balance of mitochondrial fusion and fission in a healthy cell. However, this balance is disrupted under stress conditions, resulting in apoptosis. 31 In the current study, we found that the mi- This study has several limitations. First, in addition to Drp1 and Mfn2, whether haemin can affect other proteins related to mitochondrial dynamics has not been determined. Second, we only examined the survival of haemin-pretreated BM-MSCs at 4 weeks after transplantation; therefore, long-term cell survival needs to be examined in future studies. Third, the potential mechanisms behind HO-1 regulation of mitochondrial dynamics remain unclear. Haemin contains iron, which is released by HO activity, regulating the expression of various proteins. As mitochondria are the major iron handling organelles, whether haemin regulates mitochondrial dynamics via iron requires further investigation. Fourth, as SD/H enhances endogenous HO-1 expression level, it therefore would make scientific sense to silence basal HO-1 levels to verify our study.
In summary, our results demonstrated that haemin pre-treatment, via up-regulation of HO-1 levels, significantly enhanced BM-MSC survival under ischaemic conditions by inhibiting mitochondrial fission, thus improving the therapeutic effects for treating MI. Our study shows pharmacological pre-treatment modulating the HO-1 pathway as a novel approach for enhancing MSC-based therapy for cardiovascular diseases.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.