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Angiogenesis

, Volume 21, Issue 3, pp 599–615 | Cite as

BI1 is associated with microvascular protection in cardiac ischemia reperfusion injury via repressing Syk–Nox2–Drp1-mitochondrial fission pathways

  • Hao ZhouEmail author
  • Chen Shi
  • Shunying Hu
  • Hong Zhu
  • Jun RenEmail author
  • Yundai ChenEmail author
Original Paper

Abstract

Background

Mitochondrial fission has been identified as the pathogenesis underlying the development of cardiac microvascular ischemia reperfusion (IR) injury, although the regulatory signaling upstream from fission is far from clear. Bax inhibitor is a novel anti-apoptotic factor, and, however, its role of cardiac microvascular IR injury and mitochondrial homeostasis remains unclear.

Methods

The cardiac microvascular IR injury was performed in WT mice and BI1 transgenic (BITG) mice. The alterations of microvascular structure and function were detected via electron microscope, immunohistochemistry and immunofluorescence in vivo. Cardiac microvascular endothelial cells were isolated form WT and BITG mice and underwent hypoxia/reoxygenation injury in vitro. Cellular viability and apoptosis were analyzed via MTT assay and caspase-3 activity. Mitochondrial function, morphology and apoptosis were detected. Signaling pathways were analyzed via inhibitor, siRNA and mutant plasmid.

Results

Herein, we demonstrated that Bax inhibitor 1 (BI1) was downregulated following cardiac microvascular IR injury, and its expression correlated negatively with microvascular collapse, endothelial cell apoptosis and mitochondrial damage. However, compared to wild-type mice, BI1 transgenic mice were actually protected from the acute microvascular injury and mitochondrial dysfunction. Functional studies illustrated that reintroduced BI1 directly interacted with and inhibited the Syk pathway, leading to the inactivation of Nox2. Subsequently, less Nox2 was associated with ROS downregulation, inhibiting Drp1 phosphorylated activation. Through repression of the Syk–Nox2–Drp1 signaling axis, BI1 strongly disrupted mitochondrial fission, abolishing mitochondrial apoptosis and thus sustaining endothelial cell viability.

Conclusions

In summary, our report illustrates that BI1 functions as a novel microvascular guardian in cardiac IR injury that operates via inhibition of the Syk–Nox2–Drp1-mitochondrial fission signaling axis. Thus, novel therapeutic strategies to regulate the balance between BI1 and mitochondrial fission could provide a survival advantage to microvasculature following IR stress.

Keywords

Microvascular IR injury BI1 Mitochondrial fission Syk–Nox2 signaling pathways Cardiac microvascular endothelial cells 

Introduction

A timely reperfusion strategy is the standard treatment for patients with acute myocardial infarction (AMI). However, reperfusion after ischemia always evokes ischemia reperfusion (IR) heart injury after revascularization of the occluded coronary [1]. Over the past few decades, most studies have primarily focused on the consequences of cardiomyocyte death in response to IR injury, and thus, little attention has been paid to the alterations of cardiac microvasculature in the IR injury setting [2]. Our previous clinical studies have clearly demonstrated that microvascular IR injury is an independent risk of the mortality and rehospitalization rates in patients with reperfusion therapy [3, 4]. Therefore, understanding the cellular and molecular mechanisms of microvascular IR injury may pave the road to new treatment modalities [5], which are desperately needed for the treatment of cardiac IR injury in clinical practice.

We have previously conducted numerous studies on the role of mitochondrial homeostasis in microvascular IR injury. We confirmed that FUNDC1-required mitophagy [6, 7], HK2/VDAC1/mPTP/Parkin pathways [8], calcium overload-XO-mitochondrial oxidative stress [9, 10] and Mff-mediated mitochondrial fission [11] are vital for microvascular integrity and microendothelial viability. Recent studies further confirmed that mitochondrial health is highly regulated by endoplasmic reticulum (ER) [12, 13]. Notably, an increasing number of studies from several researchers have also confirmed that ER-mitochondrial contact is considered the upstream signaling factor for mitochondrial damage [14], although the mechanism underlying this phenomenon is far from clear. Bax inhibitor-1 (BI1) is the anti-apoptotic factor responsible for mitochondrial apoptosis via mediating antagonistic action on Bax and maintaining mitochondrial outer membrane integrity [15]. Interestingly, BI1 contains six transmembrane regions and localizes to ER membranes, and it was originally identified as an inhibitor of ER stress-induced apoptosis. In addition, BI1 is also involved in the modulation of ER calcium homeostasis [16], autophagy [17], reactive oxygen species (ROS) production [18], cytosolic acidification [19] and other cellular activities [20], thus implying that it regulates an evolutionarily conserved cytoprotective pathway. However, the role of BI1 in mitochondrial homeostasis under cardiac microvascular IR injury remains elusive.

The primary role of mitochondrion is to produce and regulate the production of energy-rich molecules, such as ATP, via aerobic respiration [21]. At first glance, these cellular power plants might not appear to be of particular importance to endothelial cells, a type of cell that usually covers more than two-thirds of its energy demands by anaerobic glycolysis [22], but at second glance, mitochondria are essential for endothelial function in many ways (proliferation, apoptosis and migration) [23, 24] and are far more than just a source of ATP. Therefore, endothelial mitochondria require less respiration but more integration. Our previous study has identified that mitochondrial fission occurs after microcirculation reperfusion and induces HK2 disassociation, mPTP opening, cardiolipin oxidation and caspase-9 apoptosis in the microvascular endothelium [7, 8, 11]. However, the upstream signaling accounts for mitochondrial fission in microvascular IR injury remains unknown. Several evidences have reported that mitochondrial fission could be governed by cellular oxidative stress and/or calcium overload [25, 26, 27]. Considering the beneficial effects of BI1 in ROS production, Bax suppression and calcium balance, we wanted to determine whether BI1 is implicated in mitochondrial homeostasis via the regulation of mitochondrial fission. In this study, through gain-of-function assays on BI1 in vivo and in vitro, we confirmed that BI1 is indispensably connected to microvascular protection in response to cardiac IR injury via sustaining mitochondrial function by limiting excessive mitochondrial fission.

Materials and methods

Cardiac ischemia reperfusion injury (IR injury) model in vivo

All protocols were approved by the University of Wyoming Animal Care and Use Committee. The generation of BI1 transgenic (BI1TG) mice was reported previously [28]. Briefly, fertilized mouse eggs were flushed from the oviducts of superovulated C57BL/6 mice and pronuclei were injected with pEF HA/BI1 DNA (2 ng/μl) that had been linearized with PI–SceI at its cognate restriction site located in the pEF HA vector. The injected eggs were reimplanted in the oviducts of pseudo-pregnant imprint control region (ICR) recipient females. At 3 weeks of age, the animals were tested for the presence of the transgene by PCR analysis of their genomic DNA. Then, the wild-type (WT) mice, BI1TG mice (12 weeks old, male, n = 6/group) underwent the IR injury model. The model was conducted in vivo via an 8.0 surgical suture ligation of left anterior descending coronary artery for about 45 min to induce the ischemia damage. Then, the slipknot was loosened for about 0–24 h to cause the reperfusion injury. After the IR injury, the blood was collected and analyzed via ELISA about the CK-MB, troponin T and LDH according to our previous study [8]. Echocardiography was performed in all mice after the reperfusion according to our previous studies [29].

The gelatin-ink staining

The gelatin-ink staining was used to observe the patency of microvasculature according to our previous study [11]. Firstly, once the IR injury was completed, the gelatin-ink (3% gelatin and ink) was injected into the heart via jugular vein at the room temperature of 30 °C. Then, the hearts were cut and maintained at 4 °C for about 1 h. Finally, after 4% paraformaldehyde fixation, cryosectioning was carried out and observed under microscope. Experiments were repeated three times, and at least 10 randomly selected fields were observed.

Hypoxia reoxygenation injury (HR injury) model in vitro

The cardiac microvascular endothelial cells (CMECs) were isolated from WT mice and BI1TG mice according to our previous study [10], and CMECs were used to induce the hypoxia reoxygenation injury model in vitro. The purity of the cultured cells was assessed by performing CD31 staining and assessing the uptake of acetylated low-density lipoprotein. The hypoxia preconditioning was performed as cells cultured in a tri-gas incubator for with N2 concentration in 95% and CO2 concentration at 5% for about 45 min [30]. Then, cells were under normal culture condition for about 6 h to induce the reperfusion injury. To identify the role of ROS in mitochondrial fission, N-acetyl-l-cysteine (NAC), a cell-permeable antioxidant (10 mM), and exogenous H2O2 (0.2 nM) were introduced as negative and positive controls, respectively. To inhibit the Syk pathways, R406 (2 μM for 2 h; Selleckchem, S2194) was used to incubate with CMEC for about 2 h.

Detection of CMEC permeability and transendothelial electrical resistance (TER)

A FITC-dextran clearance assay was performed to observe changes in CMEC permeability [11]. Cells were incubated with FITC-dextran (final concentration: 1 mg/ml), allowing it to permeate through the cell monolayer. Two hours later, the FITC content remaining in the plate was measured using a fluorescent plate reader (Bio-Rad, USA) to detect the extent of permeability. TER is a measure of the ionic conductance of endothelial cells and is used to assess junctional function. TER decreases when endothelial cells retract or lose adhesion. Using an in vitro vascular permeability assay kit (ECM640, Millipore, USA), CMECs were seeded onto collagen-coated inserts at a density of 100,000 cells/insert. After reaching confluence, an electrical endothelial resistance system (Millipore, USA) was used to measure TER as previously described [31]. Experiments were repeated three times.

Western blots

Samples were lysed in ice-cold RIPA buffer supplemented with a cocktail of protease. Then, the samples were centrifuged at 14,000 rpm for 20 min at 4 °C. Equal amounts of protein were separated via SDS-PAGE and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). The 5% bovine serum albumin was used to block the samples for 1 h at room temperature [32]. Then, the membranes were incubated overnight at 4 °C with primary antibodies: Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase-9 (1:1000, Cell Signaling Technology, #9504), pro-caspase-3 (1:1000, Abcam, #ab13847), cleaved caspase-3 (1:1000, Abcam, #ab49822), c-IAP (1:1000, Cell Signaling Technology, #4952), Drp 1 (1:1000, Abcam, #ab56788), Fis 1 (1:1000, Abcam, #ab71498), Opa 1 (1:1000, Abcam, #ab42364), Mfn 1 (1:1000, Abcam, #ab57602), p-eNOS (Ser1177) (1:1000, Abcam, #ab184154), Mff (1:1000, Cell Signaling Technology, #86668), p-eNOS (Ser1117) (1:1000, Abcam, #ab184154), ET-1 (1:1000, Abcam, #ab2786), BI1 (1:1000, Abcam, #ab18852), Nox2 (1:1000, Abcam, #ab80508), p-p47phox Antibody (1:1000, Abcam, # ab74095), p-Syk (Tyr525/526) (1:1000, Cell Signaling Technology, #2711), Syk (1:1000, Cell Signaling Technology, #2712). Then the membranes were incubated with secondary antibodies at room temperature for 1 h. Band intensity was quantified using the Image-Pro Plus 6.0 software [33]. Experiments were repeated three times.

MTT, caspase-3 activity and TUNEL assay

The MTT assay was used to detect the cellular viability. In brief, MTT solution (5 mg/ml for about 20 μl) was added into the medium for about 4 h. Then, the supernatant was discarded and 100 μl DMSO was applied into the culture for about 30 min. Then, the optical density value of A490 nm (OD) was measured. The caspase-3 activity and TUNEL assay were detected to reflect the cellular apoptosis according to our previous study [34]. The level of caspase-3 activity was expressed as a percentage of the control group. The TUNEL-positive cells were imaged and counted [35], and at least 15 microvascular lumens in at least 5 randomly selected fields were observed in vivo.

Immunofluorescence and immunohistochemistry

Tissues and cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.5% Triton X-100 for 10 min. Then, the 5% normal goat serum was used to block the sample for 1 h at room temperature [36]. Then, the samples were incubated with primary antibodies overnight at 4 °C [37]. After extensively washed, samples were observed under an Axio Observer Z1 microscope [38]. The primary antibodies used in the present study were as follows: p-eNOS (Ser1177) (1:1000, Abcam, #ab184154), Syk (1:1000, Cell Signaling Technology, #2712), BI1 (1:1000, Abcam, #ab18852), VE-cadherin (1:1000, Abcam, #ab205336), F4/80 (1:1000, Abcam, #ab16911) and troponin T (1:1000, Abcam, #ab8295). Experiments were repeated three times. In vivo, at least 15 microvascular lumens in at least 5 randomly selected fields were observed. In vitro, at least 30 cells in at least 5 randomly selected fields were observed.

Syk mutant vector construction and transfection

Transfection of plasmids was performed using Turbofect (Thermo Scientific, MA, USA) or Attractene transfection reagent (Qiagen, Valencia, CA). The constitutively active form Syk (c.a.Syk) which serine at 525 site was replaced with aspartic acid (permanent phosphorylation of 525 site). For lentiviral packaging, the three PCR-amplified mutant Syk were cloned into pCMV vectors. Then, the above vectors were triple-transfected into 293T cells using Lipofectamine 2000. After transfection for 48 h, the supernatant fraction containing lentiviral particles was collected. Following amplification, the supernatant was acquired and filtered and then applied to infect CMECs as previously described [39].

To overexpress Nox2 in CMEC, the pDC316-mCMV-Nox2 adenovirus plasmid (OE-Nox2) and control adenovirus plasmid (OE-ctrl) were purchased from Vigene Biosciences. The OE-Nox2 and OE-ctrl were used to infect the CMEC.

Quantitative RT-PCR analysis

Quantitative PCR analyses were carried out using cDNAs isolated from cellular samples and primer pairs [40, 41] for MMP9 (forward, 5′-GAGAGACGTCTGGTAGATCG-3′; reverse, 5′-GTGCCAGCATGTGTCGTAGT-3′); TNFα (forward, 5′-AGATGGAGCAACCTAAGGTC-3′; reverse, 5′GCAGACCTCGCTGTTCTAGC-3′), IL6 (forward, 5′-CAGACTCGCGCCTCTAAGGAGT3′; reverse, 5′-GATAGCCGATCCGTCGAA-3′), MCP1 (forward, 5′-GGATGGATTGCACAGCCATT-3′; reverse, 5′-GCGCCGACTCAGAGGTGT-3′), ICAM1 (forward, 5′-GAGACGCAGAGGACCTTAACAG-3′; reverse, 5′-GACGCCGCTCAGAAGAACC-3′) and VCAM1 (forward, 5′-ACACCGTCATTATCTCCTG-3′; reverse, 5′-TTAGATTCACACTCGTATATGC-3′) and GAPDH (forward, 5′-AATGGTGAAGGTCGGTGTG-3′; reverse, 5′-GTGGAGTCATACTGGAACATGTAG-3′). Experiments were repeated three times.

EM analysis of microvascular

The microvascular ultrastructure in heart was evaluated using electron photomicrographs (EM) [42]. Heart samples were fixed in 5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 0.05% CaCl2 for 24 h. After washing in 0.1 M sodium cacodylate buffer, tissues were post-fixed in 1% OsO4 and 0.1 M cacodylate buffer overnight, dehydrated and embedded in Embed-812 resin [43]. The sections were stained with 2% uranyl acetate followed by 0.4% lead citrate and viewed with a Philips 400 electron microscope (Electron Microscopy Sciences). At least 15 microvascular lumens in at least 5 randomly selected fields were observed.

Transwell assay

The transwell assay was used to evaluate the migration of endothelial cells. Firstly, the endothelial cells were cultured under hypoxia/reoxygenation (HR) injury. Then, these cells were digested and added into the 24-well transwell plate for about 24 h under the normal culture condition [44]. Subsequently, the extra liquids in the upper and lower chambers were discarded. After wiping of the cells in the upper chamber, the cells in the lower chambers were fixed via pre-cooled formalin and stained by 1% crystal violet for 10 min [45]. At last, photographs were taken under the microscope and observation results were recorded. Experiments were repeated three times.

Mitochondrial membrane potential (ΔΨm), mPTP opening and ROS detection

The mitochondrial transmembrane potential was analyzed using a JC-1 Kit (Beyotime, China). Images were captured using a fluorescence microscope (OLYMPUS DX51; Olympus, Tokyo, Japan) and were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD) to obtain the mean densities of the region of interest, which was normalized to that of the control group.

The opening of the mPTP was visualized as a rapid dissipation of tetramethylrhodamine ethyl ester fluorescence. Arbitrary mPTP opening time was determined as the time when tetramethylrhodamine ethyl ester fluorescence intensity decreased by half between initial and residual fluorescence intensity according to previous study. The intracellular reactive oxygen species (ROS) generation was detected via DCFH-DA (Beyotime, Shanghai, China) through flow cytometry analysis or inverted phase contrast microscope as our previously described [46]. Experiments were repeated three times.

Coimmunoprecipitation

Coimmunoprecipitation experiments were performed as our previous study described [8]. Proteins from CMEC were cross-linked in 1% paraformaldehyde followed by washing in PBS containing 100 mmol/l glycine. The immunoprecipitates were loaded on SDS-PAGE and probed with BI1 antibody. Experiments were repeated three times.

Statistical analysis

All analyses were performed with SPSS software version 20.0 (SPSS Inc.; IBM Corp., Armonk, NY, USA). All results are expressed as the mean ± standard error of mean (SEM), and statistical significance for each variable was estimated by a one-way analysis of variance followed by Tukey’s test for the post hoc analysis. P < 0.05 was considered to indicate a statistically significant difference.

Results

BI1 is downregulated after cardiac IR injury and contributes to microvascular damage

First, we observed changes in BI1 before and after cardiac IR injury. qPCR (Fig. 1a) and western blot analysis (Fig. 1b, c) showed that the expression of BI1 progressively decreased as the reperfusion time extended (0–24 h). After reperfusion for approximately 6 h, the BI1 content was lowest. Similar results were also observed in cardiac microvascular endothelial cells (CMECs) isolated from WT mice undergoing 45 min of hypoxia and 0–24 h of reoxygenation in vitro (Fig. 1d, e). Therefore, 45 min of ischemia and 6 h of reperfusion were used in the subsequent studies.
Fig. 1

BI1 was downregulated in response to the IR injury in the infarcted area. WT mice underwent the 45-min ischemia and 0–24-h reperfusion (IR injury, n = 6/group). Subsequently, hearts were obtained and infarcted tissues were isolated. Then, these tissues were used to perform the western blots, immunohistochemistry assay and electron microscope analysis. a The transcription of BI1 in reperfused hearts. b, c The expression of BI1 was detected via western blots. d, e The CMECs were isolated from WT mice and underwent the HR injury. BI1 expression was detected via western blots. f, g Immunohistochemistry of BI1 in WT and BI1TG mice under IR injury and at least 15 microvascular lumens in at least 5 randomly selected fields were observed per group. The boxed area (microvascular lumens) under each micrograph represents the amplification of the black square. hj The cardiac damage markers were measured via ELISA. km The cardiac function in reperfused hearts was evaluated. n Gelatin-ink vascular imaging was used to detect the microvascular perfusion defect. o The HE staining of erythrocyte aggregation secondary to the vasodilation imbalance. BI1 alleviated the erythrocyte accumulation. p The change in the microvascular ultrastructure by EM in response to IR. IR injury induced microvascular wall destruction and luminal stenosis. These changes were reversed by the reintroduction of BI1. q, r TUNEL assay for the apoptotic endothelial cells and at least 15 microvascular lumens in at least 5 randomly selected fields were observed per group. s, t Cardiac microvascular endothelial cells (CMECs) were isolated from WT and BI1TG mice. And then, CMEC underwent 45-min hypoxia and 6-h reoxygenation in vitro. Cellular viability was detected via MTT assay and caspase-3 activity. #P < 0.05 versus the sham group or control group; *P < 0.05 versus IR + WT group or HR + WT cell group

Next, to explore the detailed role of BI1 in cardiac microvascular IR injury, BI1 transgenic (BI1TG) mice were used. The immunofluorescence detection of BI1 showed that BI1 expression was maintained better in BI1TG mice than WT mice during cardiac IR injury (Fig. 1f, g). The cardiac damage markers (Fig. 1h–j) and cardiac function parameters (Fig. 1k–m) also indicated that reintroduction of BI1 alleviated the acute cardiac IR injury.

To observe changes in microvascular structure with BI1 overexpression, we used gelatin-ink to fill the vessels. Once microcirculation was blocked, little gelatin-ink was detected, thus reflecting the microvascular patency. We found that IR injury seriously reduced the density of gelatin-ink, indicative of microvascular blockage (Fig. 1n). However, BI1TG mice could keep the vessels open. These data illustrated that BI1 was necessary for microvascular patency. As the consequence of blood blockade, the morphologies of red blood cells were shifted from “parachute” or “arrow” to “swollen” or “massed” via H&E staining (Fig. 1o). This may be resulted from the stoppage of turbulent blood flow or a secondary effect due to capillary blockage by other cells, such as leukocytes. However, red blood cells in BI1TG mice exhibited regular shapes. Furthermore, electron microscopy (EM) was performed to observe changes in microvascular lumen. Irregular endothelial swelling and luminal stenosis were identified in cardiac microvessels following IR injury in WT mice (Fig. 1p). However, the surfaces of the cardiac microvessels were smooth and well integrated in BI1TG mice. Finally, the TUNEL assay was carried out to measure endothelial damage. The number of TUNEL-positive CMECs was increased in WT mice and decreased in BI1TG mice during IR injury (Fig. 1q, r).

To provide more solid evidence for the beneficial effects of BI1 in endothelial cells, CMECs were isolated from WT and BI1TG mice and then analyzed during HR injury in vitro. First, HR reduced cell viability, which was recused via BI1 overexpression, as determined by the MTT (Fig. 1s) and caspase-3 activity assay (Fig. 1t). These data illustrated that IR evoked microvascular collapse and microendothelial apoptosis; however; BI1 overexpression offered beneficial influences on the microvascular structure and endothelial survival in IR injury.

Reintroduction of BI1 reverses microvascular and endothelial function

The function of microcirculation is to regulate blood flow via vasodilatation. However, IR injury significantly reduced p-eNOS expression, as evidenced by immunofluorescence (Fig. 2a, b) and western blots (Fig. 2c, d). In contrast, endothelin-1 (ET-1), a vasoconstrictor, was largely increased, as demonstrated by western blots (Fig. 2e). However, regaining BI1 upregulated p-eNOS expression and downregulated the ET-1 content. Similar results about endothelial barrier dysfunction and hyper-permeability were also obtained in CMECs isolated from WT mice but not from BI1TG mice via FITC-dextran clearance and transendothelial electrical resistance (TER) assay. FITC-dextran was applied on top of the inserts and allowed to permeate through cell monolayers. The increased endothelial permeability resulted in the retention of more FITC-dextran. Thus, FITC content remaining in the plate indicates the extent of CMEC permeability (Fig. 2f). TER assay was performed to detect CMEC barrier function. TER increases when endothelial cells adhere and spread out and decreases when endothelial cells retract or lose adhesion, reflecting endothelial barrier integrity (Fig. 2g). In vivo, immunofluorescence assay of VE-cadherin was used to observe the microvascular barrier.
Fig. 2

BI1 maintained the microvascular function. a, b The p-eNOS expression was detected via immunohistochemistry. At least 15 microvascular lumens in at least 5 randomly selected fields were observed per group. The boxed area (microvascular lumens) under each micrograph represents the amplification of the black square. ce Western blots were used to analyze the expression of p-eNOS and ET-1. f FITC-dextran clearance was measured to assess changes in endothelial permeability. FITC-dextran was applied on top of the inserts and allowed to permeate through cell monolayers. The increased endothelial permeability resulted in the retention of more FITC-dextran. Thus, FITC content remaining in the plate indicates the extent of CMEC permeability. g TER assay was performed to detect CMEC barrier function during mitochondrial fission. TER increases when endothelial cells adhere and spread out and decreases when endothelial cells retract or lose adhesion, reflecting endothelial barrier integrity. h, i The expression of VE-cadherin was reduced under IR injury, suggestive of damaged endothelial barriers. j, k The transcriptional alterations of ICAM1 and VCAM1. l, m Because of the broken endothelial barriers, there were more F4/80+ inflammatory cell infiltration into the myocardial tissue under IR injury, while the reintroduction of BI1 alleviated the inflammatory response. np The qPCR assay was applied to evaluate the changes in endothelial inflammatory markers. q, r CMEC isolated from WT or BI1TG mice were used to detect the influence of IR injury on endothelial migration response via transwell assay. #P < 0.05 versus the sham group or control group; *P < 0.05 versus IR + WT group or HR + WT cell group

IR injury markedly decreased the expression of VE-cadherin, which was restored by the regaining of BI1 (Fig. 2h, i). Additionally, IR increased intercellular adhesion molecule 1 (ICAM1) (Fig. 2j) and vascular cell adhesion protein 1 (VCAM1) transcription (Fig. 2k), but reintroduction of BI1 reduced the contents of adhesive proteins. The upregulated adhesive factor on the surface of endothelium was accompanied with more F4/80+ neutrophil permeation from blood into myocardial tissue (Fig. 2l, m). Moreover, increased transcription of inflammatory factors including TNFα, IL6 and MCP1 was also identified in reperfused heart isolated from WT mice rather than from BI1TG mice (Fig. 2n–p). However, these tendencies were recused by BI1 overexpression. Altogether, these data indicated that IR injury induced the endothelial barrier dysfunction, resulting in the activation of post-infarction inflammation response, and that BI1 overexpression rendered endothelial cells unresponsive to IR injury.

Apart from cellular viability, endothelial migration is vital for revascularization in the infarcted heart. The transwell assay results showed that HR repressed the migratory responses of CMECs (Fig. 2q, r), and these changes were improved in BI1TG cells. Altogether, this information demonstrates that BI1 is required for the microvasculature and endothelial homeostasis in the cardiac IR injury setting.

BI1 protects CMECs against HR damage via suppressing mitochondrial fission

To explain the beneficial effects of BI1 on endothelial homeostasis, we focused on cellular apoptosis, especially mitochondrial apoptosis. First, we confirmed that BI1 could sustain mitochondrial potential (Fig. 3a, c), reduce cellular ROS (Fig. 3b, d) and block excessive mPTP opening (Fig. 3e, f) during HR attack. Through western blot analysis, we confirmed that HR upregulated the contents of mitochondrial pro-apoptotic proteins and downregulated the expression of anti-apoptotic factors (Fig. 3g–n), the effects of which were reversed in BI1TG cells. These data indicated that BI1 protects CMECs against HR-mediated mitochondrial apoptosis.
Fig. 3

BI1 controlled the mitochondrial apoptosis in CMECs. a, c Mitochondrial potential was observed via the JC1 staining. b, d The cellular oxidative stress was detected via ROS staining. e, f The mPTP opening time. gn Western blots was used to analyze the change in mitochondrial apoptotic proteins. o, p Mitochondrial morphology was observed via immunofluorescence. The yellow arrows indicate the fragmented mitochondria. The mitochondrial length was measured. q Caspase-9 activity was detected to investigate the role of mitochondrial fission in mitochondrial apoptosis. #P < 0.05 versus control group; *P < 0.05 versus HR + WT cell group; @P < 0.05 versus HR + BI1TG cell group

To investigate the mechanism by which BI1 protects mitochondria against HR injury, we focused on mitochondrial fission [11], which occurs at the early stage of mitochondrial apoptosis based on our previous study. Using immunofluorescence analysis, we demonstrated that HR promoted rod-shaped mitochondrial division into several round fragmentations. In contrast, BI1 overexpression limited the formation of mitochondrial debris (Fig. 3o). Subsequently, we measured the average length of mitochondria to quantify mitochondrial fission. The average length of mitochondria after HR was approximately 2.4 ± 0.5 μm (Fig. 3p). However, BI1 overexpression reversed the length of mitochondria to 7.8 ± 1.2 μm. Then, gain- and loss-of-function assays of mitochondrial fission were conducted via FCCP and mdivi1, respectively. The fission activator FCCP was applied to BI1TG cells. In contrast, the blocker of fission mdivi1 was used in WT cells. Reactivation of fission in BI1TG cells during HR injury reincreased the number of mitochondrial fragmentations, while the average length of mitochondria was decreased again (Fig. 3o, p). In contrast, during the inhibition of fission in WT cells under HR, mitochondrial debris fragmentation was decreased, while the average length of mitochondria was increased (Fig. 3o, p). Subsequently, caspase-9 activity was measured to confirm whether BI1 sustains endothelial survival via suppressing mitochondrial fission. During HR injury, caspase-9 activity was increased in WT cells and decreased in BI1TG cells. However, FCCP treatment re-elevated caspase-9 activity in BI1TG cells (Fig. 3q). By comparison, mdivi1 reduced caspase-9 activity in WT cells. Altogether, these data suggested that BI1 provides survival advantages for CMECs via limiting mitochondrial fission.

Regaining BI1 inactivates Drp1 via a ROS-clearing effect by targeting Nox2

Mitochondrial fission is mainly regulated by Drp1 translocation from the cytoplasm to the mitochondria, where it encircles and then contracts mitochondria into several daughter mitochondria. Accordingly, we wanted to determine whether BI1 inactivates mitochondrial fission via modifying Drp1 activity. Compared to WT cells under HR stimulation, BI1 reduced mitochondrial Drp1 expression and thus reversed the expression of cytoplasmic Drp1 (Fig. 4a–c). Furthermore, Drp1 migration from the cytoplasm to mitochondria is governed by mainly its posttranscriptional modification at Ser616. Interestingly, HR promoted the phosphorylation of Drp1 at Ser616 in WT cells, the effect of which was nullified by BI1 overexpression (Fig. 4a–d). These data validated our hypothesis that BI1 is the upstream inhibitory signal for mitochondrial fission via the modulation of Drp1 at the posttranscriptional level.
Fig. 4

BI1 regulated mitochondrial fission via reducing Nox2-mediated Drp1 posttranscriptional modification. ah Western blots was used to detect the proteins’ alterations related to mitochondrial fission and mitochondrial fusion. il Induction of cellular ROS recaused the Drp1 activation in BI1TG cells. In contrast, alleviation of ROS via NAC repressed the Drp1 activation in WT cell. mo The changes in Nox2 in BI1TG cell. p, q Mitochondrial morphology was observed via immunofluorescence. The yellow arrows indicate the fragmented mitochondria. The mitochondrial length was measured. r Caspase-9 activity was detected to investigate the role of Nox2 in mitochondrial apoptosis. #P < 0.05 versus control group; *P < 0.05 versus HR + WT cell group; @P < 0.05 versus HR + BI1TG cell group

To provide more solid evidence for the regulatory role of BI1 in mitochondrial fission, fission-related proteins and fusion-involved factors were measured. In WT cells, HR elevated the expression of Mff and Fis1, the Drp1 receptors located on the surface of mitochondria (Fig. 4a–f). In contrast, BI1TG cells expressed less Mff and Fis1. Regarding mitochondrial fusion, compared to that in WT cells under HR attack, BI1TG cells contained more Mfn1 and Opa1, anti-fission elements, and thus limiting mitochondrial fission (Fig. 4a–h). This information illustrates that mitochondrial fission is launched by HR via Drp1 phosphorylated activation and that BI1 overexpression suppresses fission via inhibiting Drp1.

Our previous findings indicated that Drp1 phosphorylation is primarily regulated by ROS, calcium overload and AMPK pathways [8]. In this study, we demonstrated that NAC, an antioxidant, reduced Drp1 phosphorylation at Ser616 in WT cells under HR attack (Fig. 4i–l). In contrast, in BI1TG cells, the application of H2O2 reinduced Drp1 phosphorylation, which was accompanied with more Drp1 expression in mitochondria (Fig. 4i–l).

Given that BI1 had the ability to repress cellular oxidation via modulating the nicotinamide adenine dinucleotide phosphate oxidase (Nox) family [47], combined that Nox2 are abundant in CMECs as per our previous findings [48], we speculated whether BI1 affects mitochondrial fission via Nox2. As shown in Fig. 4m–o, the protein expression of Nox2 was dramatically increased in WT cells but not in BI1TG cells under HR treatment (Fig. 4m–o). Meanwhile, the phosphorylation of p47phox, an activator of Nox2 activities, was also increased in WT cells but not in BI1TG cells during HR attack (Fig. 4m–o). These data indicated that Nox2 was regulated by HR via BI1. To investigate whether Nox2 is responsible for mitochondrial fission, Nox2 was overexpressed in BI1TG cells (Fig. 4m–o). Overexpression of Nox2 obviously increased the number of mitochondrial debris and thus reduced the mitochondrial length in BI1TG cells (Fig. 4p, q). Moreover, overexpression of Nox2 also reincreased the caspase-9 activity in BI1TG cells (Fig. 4r).

BI1 regulates the Nox2 via the Syk pathway

Our previous studies reported that Nox2 could be regulated by cAMP/PKA/Rho pathways in CMECs under high-glucose stimulation [48]. Recently, spleen tyrosine kinase (Syk) was identified as the novel signaling molecule responsible for Nox-involved ROS production. Reduced ROS generation in Nox2-deficient platelets is associated with impaired activation of Syk [49]. Accordingly, we wondered whether Syk is associated with the Nox2 inhibition induced by BI1. First, we demonstrated that Syk was significantly increased in response to HR injury as evidenced by elevated phosphorylated Syk (Fig. 5a, b), and these processes were controlled by BI1 because BI1 overexpression reduced HR-mediated Syk activation.
Fig. 5

BI1 reduced the Syk–Nox signaling pathways. ad Syk activation was detected via western blots. R406, the inhibitor of Syk was used to block the Syk phosphorylated activation in WT cell. In contrast, the mutant Syk, c.a.Syk, was used to enhance the Syk phosphorylated activation in BI1TG cell. e Co-IP was used to detect the interaction between p-Syk and BI1. f Confocal microscopy was used to observe the proteins interaction between Syk and BI1. #P < 0.05 versus control group; *P < 0.05 versus HR + WT cell group; @P < 0.05 versus HR + BI1TG cell group

To establish the role of Syk in Nox2, the Syk inhibitor R406 was used. Compared to WT cells under HR treatment, application of R406 attenuated the expression of p-Syk (Fig. 5a, b), which was accompanied with downregulated Nox2 and phos-p47phox expression (Fig. 5a–d). Subsequently, a gain-of-function assay of Syk was carried out via Syk mutant transfection in BI1TG cells. The serine at site 525 in the constitutively active form of Syk (c.a.Syk) was replaced with aspartic acid (permanent phosphorylation of site 525). After transfection with c.a.Syk in BI1TG cells, p-Syk was significantly increased, which was accompanied by elevated Nox2 and pho-p47phox expression (Fig. 5a–d). Thus, through the upregulation and downregulation assay about Syk, we reconfirmed that Syk was trigged by HR and contributed to Nox2 activation; however, reintroduction of BI1 inhibited the Syk–Nox2 signaling pathways.

Finally, to investigate the mechanism by which BI1 inactivates Syk, we conducted an immunoprecipitation (IP) assay to observe the interactions between BI1 and Syk. A direct interaction between BI1 and Syk was observed in BI1TG cells under HR attack (Fig. 5e). Furthermore, considering that BI1 often resides in the cytoplasm, we hypothesized that the BI1–Syk interaction also occurs in the cytoplasm. Indeed, confocal microscopy showed approximately 75% colocalization of BI1 and Syk in the cytoplasm of BI1TG cells (Fig. 5f). These observations explained that BI1 has the ability to directly bind and inhibit Syk activity, leading to Nox2 downregulation.

Discussion

In the current study, we first demonstrated that BI1 was downregulated in response to cardiac IR injury. Reintroduction of BI1 could enhance the resistance of cardiac microcirculation to IR injury via sustaining mitochondrial homeostasis. Functional assays demonstrated that BI1 directly interacts with and inactivates Syk. Defective Syk failed to upregulate Nox2, leading to the suppression of ROS outbursts. The inhibitory effects of BI1 on Nox2-related ROS contributed to Drp1 inactivation via posttranscriptional modifications, hampering mitochondrial fission and thus sustaining mitochondrial function. Well-structured mitochondria closed apoptosis pathways and provided a survival advantage for cardiac microvascular endothelial cells, finally improving microvascular structure and function in the IR injury setting. To our knowledge, this is the first study to introduce BI1 in a cardiac microvascular injury involving Drp1-dependent mitochondrial fission, Nox-associated oxidative stress and Syk pathways (Fig. 6).
Fig. 6

BI1 inactivates Syk–Nox2 pathways and subsequently repressed Drp1 activation, abolishing fatal mitochondrial fission. Defective mitochondrial fission promotes the survival of CMEC and preserves the microvascular structure and function

Compared to cardiomyocytes, microvasculature and microendothelial cell damage in the context of cardiac IR injury has unfortunately been a neglected topic in the past 30 years [50]. Based on our previous clinical studies, microvascular IR injury occurs in 15–50% of patients during or after PCI treatment [3, 51]. Considering the central role of microcirculation in the exchange of matter, oxygen and energy between blood and cardiomyocytes, reperfusion injury of the microvascular bed post-ischemia is a key factor contributing to secondary myocardial injury [11]. Accordingly, elucidating the mechanisms underlying microvascular IR injury would offer new potential targets to treat cardiac microvascular IR injury.

Our previous studies have explored several targets for microvascular reperfusion: Mff-required mitochondrial fission [11], Parkin-dependent mitophagy [8], FUN14 domain-containing 1 (FUNDC1)-activated platelet aggregation [6], xanthine oxidase (XO)-induced endothelial oxidative stress [10], IP3R/MCU-triggered [Ca2+]c/[Ca2+]m overload [9] and Ripk3-related mitochondrial apoptosis [7]. These findings all note that mitochondrial homeostasis, including mitochondrial fission, mitophagy and mitochondrial apoptosis, are necessary for endothelial protection in response to cardiac IR injury. However, we did not previously understand the upstream regulatory signaling of mitochondrial homeostasis, especially mitochondrial fission. In the current study, we identified BI1 as a protector of mitochondrial function in the IR injury setting. BI1 governs mitochondrial fission, sustains mitochondrial potential, represses ROS production and manages mitochondrial apoptosis. Notably, considering that BI1 is primarily located on ER, the role of BI1 in mitochondrial homeostasis hints that ER-mitochondrion contact is the key regulatory mechanism responsible for mitochondrial structure and function in cardiac microvasculature. Recent studies have identified ER-localized IP3R as the major factor of mitochondrial function (mitophagy and mitochondrial fission) via regulating ER-mitochondria calcium signaling in response to cardiomyocyte IR injury [52]. In our previous study, we also reported ER-localized XO as an apoptotic signaling massager via amplifying ER-mitochondria oxidative cascade reactions in cardiac microvascular IR injury [10]. In the current study, we described a novel element, BI1, that belongs to ER but exerts a mitochondrial protective action. This finding may add more information on the role of ER-mitochondria contact in IR injury.

We determined that BI1 protected mitochondria against IR injury via limiting excessive mitochondrial fission. Mechanistically, BI1 inhibited Drp1 via posttranscriptional phosphorylation at Ser616 [53]. Phosphorylated Drp1 at Ser616 is an active form that can translocate to the surface of mitochondria and mediate mitochondrial contraction via GTP consumption [54]. Furthermore, we demonstrated that BI1 impaired Drp1 phosphorylated activation via ROS, which was identified in a previous study as the upstream activator of fission [55]. Functional assays demonstrated that the ROS-clearing effects of BI1 were attributed to Nox2 downregulation, and the levels of BI1 were reversely associated with Nox2 expression in CMECs under HR treatment. These findings were similar to those of a previous study demonstrating BI1 to be an inhibitor of the Nox family [47]. Notably, we explained for the first time that the inhibitory role of BI1 in Nox2 is dependent on the Syk pathway. Syk is a nonreceptor tyrosine kinase best known for its role downstream of immune receptors triggering a series of signaling pathways leading to the proliferation, survival, differentiation, migration and production of reactive oxygen species and cytokines [56]. Recently, Syk was reported to play a role in kidney IR injury [57]. Genetic deletion of Syk protected murine kidneys against IR injury via reducing inflammatory response. Our data also demonstrated that Syk was increased during cardiac microvascular IR injury, and inhibition of Syk could reduce the microvascular damage. Therefore, this observation offers a potential target to intervene with microvascular IR injury. Finally, although we demonstrated that BI1 could directly interact with Nox2 in the cytoplasm, we do not yet know what BI1 protein domain is responsible for its interaction with Syk. More structural analysis of BI1 is required to answer this question. Meanwhile, whether the present findings can be applied to cardiomyocyte IR injury warrants further investigation.

Collectively, our report demonstrates a novel regulator for mitochondrial homeostasis, BI1, which is a guarantor of mitochondrial structure and function in the cardiac microvasculature. BI1 inactivated Syk–Nox2 pathways and subsequently repressed Drp1 activation, abolishing fatal mitochondrial fission and preserving microvascular structure and function. These findings open a new window for treating cardiac microvascular IR injury in the future.

Notes

Author contributions

HZ and YDC involved in conception and design, performance of experiments, data analysis and interpretation, and manuscript writing; HZ, CS and JR involved in the development of methodology, SYH and JR involved in the data acquisition, and HZ and YDC involved in financial support, study supervision and final approval of manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81770237). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors have declared that they have no conflicts of interest.

Data access

The authors are available to share their data.

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Chinese PLA General HospitalMedical School of Chinese PLABeijingChina
  2. 2.Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Radiation OncologyPeking University Cancer Hospital and InstituteBeijingChina
  3. 3.Center for Cardiovascular Research and Alternative MedicineUniversity of Wyoming College of Health SciencesLaramieUSA

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