Cardiac Ischemic Preconditioning Promotes MG53 Secretion Through H2O2-Activated Protein Kinase C-δ Signaling
Abstract
Background:
Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (mitsugumin 53; also called TRIM72), not only plays an essential role in IPC-mediated cardioprotection against ischemia/reperfusion injury but also ameliorates mechanical damage. In addition to its intracellular actions, as a myokine/cardiokine, MG53 can be secreted from the heart and skeletal muscle in response to metabolic stress. However, it is unknown whether IPC-mediated cardioprotection is causally related to MG53 secretion and, if so, what the underlying mechanism is.
Methods:
Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo, isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. Moreover, using recombinant MG53 proteins, we investigated the potential biological function of secreted MG53 in the context of IPC and ischemia/reperfusion injury.
Results:
We found that IPC triggered robust MG53 secretion in rodents in vivo, perfused hearts, and cultured cardiac myocytes without causing cell membrane leakage. Mechanistically, IPC promoted MG53 secretion through H2O2-evoked activation of protein kinase-C-δ. Specifically, IPC-induced myocardial MG53 secretion was mediated by H2O2-triggered phosphorylation of protein kinase-C-δ at Y311, which is necessary and sufficient to facilitate MG53 secretion. Functionally, systemic delivery of recombinant MG53 proteins to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice but also protected rodent hearts from ischemia/reperfusion injury even in the absence of IPC. Moreover, oxidative stress by H2O2 augmented MG53 secretion, and MG53 knockdown exacerbated H2O2-induced cell injury in human embryonic stem cell–derived cardiomyocytes, despite relatively low basal expression of MG53 in human heart.
Conclusions:
We conclude that IPC and oxidative stress can trigger MG53 secretion from the heart via an H2O2–protein kinase-C-δ–dependent mechanism and that extracellular MG53 can participate in IPC protection against cardiac ischemia/reperfusion injury.
Clinical Perspective
What Is New?
We have provided multiple lines of evidence that MG53 (mitsugumin 53)is secreted from the heart in rodents in response to ischemic preconditioning or oxidative stress and that the secreted MG53 protects the heart against ischemia/reperfusion injury.
Ischemic preconditioning–induced MG53 secretion is dependent on H2O2-evoked, Src-mediated phosphorylation of protein kinase-C-δ-Y311.
In human heart, MG53 is expressed at a level ≈1/10 of its skeletal muscle counterpart. MG53 secretion is triggered by oxidative stress in human embryonic stem cell–derived cardiomyocytes, whereas deficiency of MG53 exacerbates oxidative injury in these cells.
What Are the Clinical Implications?
Our in vivo and ex vivo results define secreted MG53 as an essential factor conveying ischemic preconditioning–induced cardioprotection.
Because systemic delivery of MG53 protein restores ischemic preconditioning–mediated cardioprotection in MG53-deficient mice, recombinant human MG53 protein can be developed into a novel treatment for various diseases of human heart in which the endogenous MG53 is low.
Introduction
Ischemic heart disease has become the most important cause of death around the world.1 Because adult mammalian cardiomyocytes are terminally differentiated cells with very limited ability for proliferation, cardiomyocyte death induced by ischemic injury and other insults leads to permanent loss of the cardiac functional unit, resulting in depressed cardiac function, arrhythmia, heart failure, and sudden death. The best way to reduce cardiac ischemic injury–induced cell loss is timely restoration of coronary blood flow, that is, reperfusion. However, myocardial reperfusion leads to further damage of the heart, called ischemia/reperfusion (I/R) injury.2–4 Ischemic preconditioning (IPC), in which nonlethal ischemic stress to the heart prevents the subsequent lethal cardiac I/R injury, is the most powerful intrinsic protection against I/R injury of the heart,5,6 as well as other organs.7–9 Although multiple mechanisms,10 including alterations in metabolism,11 mitochondrial permeability transition pore,12,13 osmotic swelling,14 and free radicals,15 have been implicated in cardiac IPC, our previous studies place MG53 (mitsugumin 53; also called TRIM72) in the central position of IPC cardioprotection. Intracellular MG53 is essential for the activation of reperfusion injury salvage kinase cardioprotective signaling, thus contributing to IPC and postconditioning protection.16,17
The heart has both mechanical and endocrine functions, and its cells—including cardiomyocytes, fibroblasts, vascular smooth muscle cells, and endothelial cells—secrete a variety of regulatory proteins in response to alterations in myocardial oxygen or nutrient supply.18–20 Proteins secreted from the heart, namely cardiokines, participate in pathological and physiological processes.21–23 For example, both ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide) are secreted from the heart in response to myocardial ischemia, which, in turn, exhibit cardiac protective effects.24–26 Recently, we demonstrated that under metabolic stress MG53 is secreted into the extracellular space and acts as a myokine/cardiokine to induce systemic insulin resistance and metabolic disorders through disrupting insulin receptor signaling.27 However, it is unclear whether MG53 secretion is sensitive to ischemia and whether secreted MG53 is functionally similar to its intracellular counterpart, conveying cardiac protective effects. Here, we show that IPC markedly induces MG53 secretion through activating H2O2–protein kinase-C-δ (PKC-δ dependent signaling and that extracellular MG53 can participate in IPC and protect the heart from I/R injury.
Methods
The data, analytical methods, and study materials are available to other researchers for purposes of reproducing the results or replicating the procedure. All materials are available in our laboratory at Peking University. Detailed methods are provided in the Data Supplement.
Animal Models
Animals were maintained in the Laboratory Animal Center (an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care) at Peking University, Beijing, China. The animals were randomly allocated to experimental groups. Male animals were used unless otherwise noted in specific experiments. We did not use noninclusion or exclusion parameters. We did not blind investigators to treatments, but no subjective assessments were made. All procedures involving experimental animals (mice and rats) were performed following protocols approved by the Committee for Animal Research of Peking University and conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 86-23, revised 2011).
The generation of mg53−/−16,28 and mg53h-TG29 mice was described previously. PKC-δ−/− mice were purchased from RIKEN BRC (catalog no. 00457). Sprague-Dawley rats (8 weeks old) were purchased from Vital River Laboratories (Beijing, China).
Isolated Rodent Heart Langendorff Perfusion and Perfusate Collection
Langendorff perfusion of the heart was performed as described previously.16,27 Briefly, adult rats (200–250 g) or mice (20–30 g) were anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg). The heart was excised and perfused on a Langendorff apparatus at a constant pressure of 55 mm Hg with Krebs-Henseleit solution (in mmol/L: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4·7H2O 1.2, KH2PO4 1.2, and glucose 11.1). The buffer was continuously gassed with 95% O2/5% CO2 (pH 7.4) and warmed by a heating bath/circulator. Heart temperature was continuously monitored and maintained at 37±0.5°C.
Global ischemia was induced by cessation of perfusion. IPC was achieved by 4 episodes of 5 minutes of ischemia followed by 5 minutes of reperfusion. Control hearts were continuously perfused (Figure 1A and Figure IA in the Data Supplement).
Figure 1. Cardiac ischemic preconditioning induces MG53 (mitsugumin 53) secretion.A, Schematic diagram showing the protocol for perfusate collection from ischemic preconditioning (IPC) perfused rodent hearts. B and C, Representative Western blots and averaged data of the MG53 from the perfusate of perfused MG53-deficient (mg53−/−; B) and MG53 heart-specific overexpression transgenic (mg53h-TG) male mouse hearts (C), together with their respective male wild-type(wt) littermates, during control (−60 to 0 minutes), 0 to 60 minutes, and 60 to 120 minutes after IPC; n=6. *P<0.05, **P<0.01 as indicated. D, Representative Western blots and averaged data of MG53 from the perfusate of perfused female mouse hearts during control (–60 to 0 minutes), and 0 to 60 or 60 to 120 minutes after IPC; n=4. *P<0.05, **P<0.01 as indicated. E, Representative Western blots and averaged data of MG53 from the perfusate of perfused female rat hearts during control (–60 to 0 minutes), and 0 to 60 or 60 to 120 minutes after IPC; n=6. **P<0.01 as indicated. F, Averaged data of the MG53 concentrations in the serum of male and female rats subjected to sham or IPC operation in vivo (male rats: n=8 for sham, 10 for IPC; female rats: n=6). *P<0.05 vs baseline. In all bar graphs, data are normalized to the corresponding nonspecific bands (NS), which were obtained through brilliant green staining, and are presented as mean±SEM.
The outlet perfusate was collected for different periods of time as indicated. For catalase treatment, PEG-catalase (300 U/mL for 10 minutes before IPC) was included in the perfusion solution. The collected perfusate samples were centrifuged at 3000 rpm with Amicon Ultra-15 10K Centrifugal Filter Devices (Millipore, catalog no. UFC801096) 3 times for 15 minutes. The concentrated perfusate samples were used for subsequent analysis. Nonspecific (total) bands were stained with brilliant green dye.
In Vivo Rodent Myocardial I/R and IPC Model
For the setup of experimental models of myocardial ischemia and IPC and the selection of the variables for the assessment of cardiac injury, we were referred to the previous guidelines.30,31 In vivo rodent I/R injury was performed as previously described.16 Specifically, Sprague-Dawley rats (200–250 g) and male mg53−/− mice (20–30 g) were anesthetized with pentobarbital (40 mg/kg IP) and ventilated through a tracheostomy on a Harvard rodent respirator. A midline sternotomy was performed, and a reversible coronary artery snare occluder was placed around the left anterior descending coronary artery. Myocardial I/R was performed by tightening the snare for 45 minutes (in rats) or 30 minutes (in mice) and then loosening it (reperfusion) for 24 hours (for protein extraction, cardiac function assessment, and infarct size measurement). Blood samples for lactate dehydrogenase (LDH) measurement were collected 10 minutes after reperfusion from rodents subjected to I/R and centrifuged for 10 minutes at 3000 rpm for serum.
For recombinant human MG53 protein (rhMG53) treatment, the rats subjected to in vivo I/R injury were treated with double intravenous injection of rhMG53/BSA (3 mg/kg, 1 injection at 5 minutes before the start of ischemia and the other at 5 minutes before the start of reperfusion); the mg53−/− mice were treated with intravenous injection of rhMG53/BSA (6 mg/kg, 40 minutes before IPC procedure).
Determination of myocardial injury by infarct size measurement and LDH release was performed as previously described.16,32 Cardiac troponin T release was assessed by the ELISA kits from Flarebio Biotech (catalog no. CSB-E16443r for rats; and catalog no. KE1753, for mice).
IPC of the in vivo rodent hearts was induced by 4 episodes of 5 minutes of ischemia followed by 5 minutes of reperfusion. Blood samples for serum MG53 measurement were collected before IPC (0 minutes) and 5/10/30 minutes after the last ischemia from rats subjected to IPC and centrifuged for 10 minutes at 3000 rpm for serum.
Rat serum MG53 was measured with an ELISA kit from Flarebio Biotech (catalog no. CSB-EL024511RA).
Adenoviral Infection of Neonatal Rat Ventricular Myocytes
Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 1-day-old Sprague-Dawley rats and implemented by adenovirus-mediated gene transfer by methods described previously.16 Adenoviral vector expressing the β-gal, PKC-δ, or PKC-δ-Y311F mutation was generated and amplified by Sinogenomax. The collected medium samples were centrifuged at 4000 rpm in Amicon Ultra-4 10K Centrifugal Filter Devices (Millipore) for 20 minutes for further Western blot detection of MG53.
Western Blots
Western blots were performed as previously described.16,27,28
Human Donor and Criteria
Normal human ventricular tissue was obtained from the US National Institutes of Health NeuroBioBank at the University of Maryland (Baltimore).
Human Embryonic Stem Cells Induced Cardiomyocytes
Human cardiomyocytes were prepared as previously described.33 Human embryonic stem (ES) cells H9 were differentiated into cardiomyocytes with a chemical-defined, xeno-free, small molecule–based method as previously reported. H9 cells were maintained on a 6-well plate precoated with Matrigel (BD Biosciences, catalog no. 354277) in E8 medium (basal medium, Life Technologies, catalog no. A1517001). When the cells reached 70% confluence, the medium was replaced with basal medium supplemented with 6 μmol/L CHIR99021 (Selleckchem, catalog no. S1263-25 mg). Forty-eight hours later, the medium was changed to basal medium supplied with 2 μmol/LWnt-C59 (Biorbyt, catalog no. orb181132) for another 48 hours. The cells were then maintained in basal medium, changing to fresh media every 48 hours. Beating cardiomyocytes emerged around day 8. On the 10th day, basal medium was replaced with RPMI 1640 without glucose (Life Technologies, catalog no. 11879020) to purify the cardiomyocytes. Two days later, the cardiomyocytes were associated with TrypLE (Life Technologies) for 10 minutes and passaged to a Matrigel-precoated well plate.
Materials
For Western blotting, the anti–total-PKC-δ antibody (catalog no. 610397; 1:1000) was from BD Biosciences. Anti–phospho-PKC-δ antibody (phosphorylation at Y311; catalog no. 2055; 1:1000), anti–phospho-Src antibody (phosphorylation at Y416; catalog no. 6943; 1:1000), anti-Src antibody (catalog no. 2109; 1:1000), and anti–cleaved caspase-3 antibody (catalog no. 9661; 1:1000) were from Cell Signaling Technology. Flag antibody (catalog no. F1804; 1:5000) was from Sigma-Aldrich. GAPDH antibody (catalog no. BE0023; 1:10000) was from Bioeasy Technology. A high-affinity custom monoclonal antibody (anti-MG53; final concentration, 0.5 μg/mL) was used to assess MG53 in heart perfusate and NRVM culture medium.
H2O2 (catalog no. 34,988-7), PEG-catalase (catalog no. C4963), (–)–isoproterenol hydrochloride (catalog no. I6504), H-89 dihydrochloride hydrate (catalog no. B1427), phorbol 12-myristate 13-acetate (catalog no. P8139), and chelerythrine chloride (catalog no. C2932) were from Sigma-Aldrich. Protein kinase A (PKA) inhibitor 14 to 22 amide (catalog no. 476485) was from Calbiochem. Go6983 (catalog no. S2911), PP1 (catalog no. S7060), and Bosutinib (SKI-606; catalog no. S1014) were from Selleck. Brilliant green (catalog no. 5141-20-8) for staining of the nonspecific (total) bands was from Amresco.
Statistical Analysis
Data are expressed as mean±SEM. Statistical analysis was performed with GraphPad Prism version 8.0.1 (GraphPad Software, Inc) and the SPSS 18.0 software package (SPSS Inc). Data groups (2 groups) with normal distributions were compared by use of the 2-sided unpaired Student t test. Comparisons between multiple groups were assessed using a 1-way ANOVA with Bonferroni correction. For grouped analyses, either multiple unpaired t tests or 2-way ANOVA with the Tukey multiple-comparisons test was performed. Death rates of rats were compared with the χ2 test. No statistical method was used to predetermine sample size.
Results
Cardiac IPC Induces MG53 Secretion
To identify IPC-sensitive myocardial secreted proteins, we performed IPC (4 episodes of 5-minute ischemia followed by 5-minute reperfusion) in isolated Langendorff-perfused rat hearts. The perfusate of IPC was collected during 0 to 30 minutes after the last episode of ischemia; the control perfusate was collected from the heart without IPC during the same period (Figure IA in the Data Supplement). After concentration, the perfusate samples were subjected to SDS-PAGE and stained with Coomassie blue. Compared with the control group, the IPC group was associated with an increase in the density of a band of ≈55 kDa (Figure IB in the Data Supplement), which was subjected to mass spectrum analysis. The repeated short-term I/R cycles of IPC did not cause cardiac injury as indexed by unaltered LDH concentration in the perfusate (Figure IIA in the Data Supplement).
In 3 independent experiments, 91 proteins with a high score (>80 defined as positive) were identified by at least 1 experiment, and 22 of them were positive in at least 2 experiments as listed in Table I in the Data Supplement (see also Figure IC in the Data Supplement). In particular, 5 proteins were positive in all of the 3 experiments (Figure IC and ID in the Data Supplement), and among them, MG53, which was previously demonstrated to mediate cardiac IPC protection intracellularly16 and identified as a myokine/cardiokine,27 was selected for further analysis.
Consistent with the mass spectrum data from isolated rat hearts, IPC profoundly increased perfusate MG53 levels in Langendorff-perfused male wild-type mouse hearts by 5.50±0.32- and 4.26±0.40-fold from baseline over 0 to 60 and 60 to 120 minutes of reperfusion, respectively (Figure 1A and 1B), without inducing myocardial membrane leakage as indexed by LDH and cardiac troponin T concentration (Figure IIB in the Data Supplement). The identity of the detected band in the perfusate was verified with the mice lacking MG53 (mg53−/−)16,28 or mice with cardiac-specific overexpression of MG53 (mg53 h-TG).29 IPC could not induce MG53 release in hearts from mg53−/− mice, whereas IPC-induced MG53 release was markedly augmented in the hearts of mg53 h-TG mice (Figure 1B and 1C) in the absence of obvious cardiac injury (Figure IIC and IID in the Data Supplement). IPC-induced MG53 secretion was also observed in the isolated hearts of female mice (Figure 1D) and rats (Figure 1E). IPC was able to increase serum MG53 concentration in both male and female rats in vivo compared with the sham group (Figure 1F) without altering serum LDH and cardiac troponin T level (Figure IIE and IIF in the Data Supplement). These data indicate that IPC can induce MG53 release from the heart in both in vivo and ex vivo models without causing cardiac damage.
H2O2 Is Required for IPC-Induced MG53 Secretion
Next, we aimed to determine the mechanism underlying the IPC-induced release of MG53. Cardiac reactive oxidative species (ROS) has been implicated in IPC signaling.34,35 H2O2, a key component of ROS, was increased in the IPC group compared with the control group (Figure 2A). Pretreatment of PEG-catalase, an H2O2 scavenger, prevented IPC-induced MG53 release in perfused rat hearts (Figure 2B). In cultured cardiomyocytes, we used repetitive short-term hypoxia/reoxygenation cycles, that is, hypoxia preconditioning, to mimic IPC in the absence of cardiomyocyte injury (Figure IIG in the Data Supplement). Similar to IPC, hypoxia preconditioning in NRVMs also augmented MG53 abundance in the culture medium, which was abolished by PEG-catalase pretreatment (Figure 2C). It is notable that short-term pretreatment (10 minutes) of NRVMs with H2O2 at concentrations ranging from 100 to 200 μmol/L protected cardiomyocytes against hypoxia/reoxygenation–induced injury, whereas prolonged treatment of H2O2 (24 hours) triggered cell death even at a concentration as low as 50 μmol/L (Figure IIH and III in the Data Supplement), indicating that a short exposure of cardiomyocytes to H2O2 is protective and sustained H2O2 treatment of cells is detrimental. Short-term treatment of H2O2 (100 µmol/L) not only protected cultured NRVMs but also induced robust MG53 release in a dose- and time-dependent manner (Figure 2D and 2E), which was blocked by PEG-catalase pretreatment (Figure 2F). These results strongly suggest that H2O2 is both necessary and sufficient for IPC-mediated MG53 release in the heart.
Figure 2. H2O2 is required for ischemic preconditioning (IPC)–induced MG53 (mitsugumin 53) secretion.A, Average data of H2O2 production in perfused rat hearts with or without IPC, ex vivo; n=4. **P<0.01 as indicated. B, Representative Western blots and averaged data of MG53 from the perfusate from perfused rat hearts during control (−60 to 0 minutes), 0 to 60 minutes, and 60 to 120 minutes after IPC with or without PEG-catalase pretreatment (300 U/mL for 10 minutes before IPC); n=5. *P<0.05 as indicated. C, Representative Western blots and averaged data of MG53 from the culture medium of neonatal rat ventricular cardiomyocytes (NRVMs) with hypoxia preconditioning (30 minutes of hypoxia followed by 30 minutes of reoxygenation, 3 cycles) treatment; n=5. **P<0.01 as indicated. D and E, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 treatment at different dosages and time points; n=5. *P<0.05, **P<0.01 as indicated. F, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 (100 µmol/L for 20 minutes) treatment with or without PEG-catalase pretreatment (500 U/mL); n=5. *P<0.05, as indicated. For B through F, data are normalized to the corresponding nonspecific bands (NS), which were obtained through brilliant green staining, and are presented as mean±SEM.
PKC, But Not PKA, Is Required for IPC-/H2O2–Induced MG53 Secretion
Previous studies have demonstrated that a wide range of serine/threonine and tyrosine protein kinases regulate cell secretion, among which PKA and PKC are the most important.36,37 Therefore, we next determined which protein kinase is responsible for MG53 secretion. Activation of cAMP-dependent PKA signaling by β-adrenergic stimulation with isoproterenol (at 10–100 µmol/L) did not elicit secretion of MG53, whereas H2O2 (100 μmol/L) induced profound MG53 secretion in cultured NRVMs (Figure 3A). In addition, inhibition of PKA with either a peptide inhibitor, PKA inhibitor 14 to 22 amide, or small-molecule inhibitor, H-89 dihydrochloride hydrate, did not affect H2O2-induced MG53 secretion (Figure 3B and 3C), suggesting that PKA is not involved in the regulation of MG53 secretion. In sharp contrast, PKC activation by phorbol 12-myristate 13-acetate overtly increased H2O2-induced MG53 secretion in a dose-dependent manner (Figure 3D), whereas inhibition of PKC with either chelerythrine chloride or Go6983 largely blocked both H2O2- and hypoxia preconditioning–induced MG53 secretion (Figure 3E through 3G). Thus, MG53 secretion from cardiomyocytes is dependent on activation of PKC rather than PKA.
Figure 3. Protein kinase-C (PKC), but not protein kinase-A (PKA), is required for ischemic preconditioning–/H2O2-induced MG53 (mitsugumin 53) secretion.A, Representative Western blots of MG53 from the culture medium of neonatal rat ventricular cardiomyocytes (NRVMs) treated with different dosages of isoproterenol (ISO) for 1 hour. The medium from NRVMs treated with H2O2 (100 µmol/L for 20 minutes) was used as positive control. B and C, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 (100 µmol/L for 20 minutes) with or without PKA inhibitor 14 to 22 amide (PKI; 5 μmol/L for 6 hours, n=4; B) or H-89 dihydrochloride hydrate (H-89; 1 μmol/L for 6 hours, n=4; C) pretreatment. D, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 (100 µmol/L for 20 minutes) with or without phorbol 12-myristate 13-acetate (PMA; 1 hour) pretreatment; n=6. *P<0.05, **P<0.01 as indicated. E and F, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 (100 µmol/L for 20 minutes) with or without chelerythrine chloride (Che; 10 μmol/L for 1 hour, n=5; E) or Go6983 (100 nmol/L for 1 hour, n=5; F) pretreatment. *P<0.05 as indicated. G, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with hypoxia preconditioning (HPC; 30 minutes of hypoxia followed by 30 minutes of reoxygenation, 3 cycles) treatment with or without Che pretreatment (10 μmol/L for 1 hour before HPC); n=5. **P<0.01 as indicated. For B through G, data are normalized to the corresponding nonspecific bands (NS), which were obtained through brilliant green staining, and are presented as mean±SEM. n.s. Indicates not significant.
IPC Triggers MG53 Secretion Through H2O2-Activated PKC-δ Signaling
The PKC family consists of ≈10 members,38 and among them, PKC-δ has been identified as one of the most important mediators of cardiac IPC protection.39 In addition, previous studies have reported that PKC-δ can be activated by ROS-related signaling.40,41 It is notable that we showed that downregulation of PKC-δ with siRNA abolished H2O2-induced MG53 secretion in NRVMs (Figure 4A and 4B). Concomitantly, deficiency of PKC-δ markedly attenuated IPC-induced MG53 secretion in perfused mouse hearts lacking PKC-δ (PKC-δ−/−; Figure 4C and Figure III in the Data Supplement), substantiating the important role of PKC-δ in IPC-induced MG53 secretion. In contrast, adenoviral gene transfer of PKC-δ exaggerated H2O2-induced MG53 secretion in NRVMs (Figure 4D and 4E). Furthermore, overexpression of PKC-δ in vivo with coronary artery delivery of adenovirus also significantly enhanced IPC-induced MG53 secretion in perfused rat hearts (Figure 4F and Figure IV in the Data Supplement).
Figure 4. Ischemic preconditioning (IPC) triggers MG53 (mitsugumin 53) secretion through H2O2-activated protein kinase-C-δ (PKC-δ) signaling.A, Representative Western blots and averaged data of PKC-δ from cultured neonatal rat ventricular cardiomyocytes (NRVMs) transfected with scrambled or 2 sets of PKC-δ siRNAs; n=4 for siRNA2, 6 for Scrambled and siRNA1. **P<0.01 vs scrambled group. B, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with H2O2 treatment (100 µmol/L for 20 minutes) transfected with scrambled or two sets of PKC-δ siRNAs; n=5. **P<0.01 as indicated. C, Representative Western blots and averaged data of MG53 from the perfusate from perfused hearts of wild-type(wt) and PKC-δ−/− mice during control (−60 to 0 minutes), 0 to 60 minutes, and 60 to 120 minutes after IPC; n=5. **P<0.01 as indicated. D, Representative Western blots and averaged data of adenovirus (Ad)-PKC-δ overexpression in NRVMs; n=4. **P<0.01 as indicated. E, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs with or without H2O2 treatment (100 µmol/L for 20 minutes) in the presence or absence of Ad-PKC-δ infection; n=5. *P<0.05 as indicated. F, Representative Western blots and averaged data of MG53 from the perfusate from perfused rat hearts during control (−60 to 0), 0 to 60 minutes, and 60 to 120 minutes after IPC with or without PKC-δ overexpression (coronary delivery of Ad-PKC-δ); n=6. *P<0.05 as indicated. G, Representative Western blots and averaged data of phosphorylated Y311 site of PKC-δ and total PKC-δ from cultured NRVMs with or without H2O2 treatment (100 µmol/L for 20 minutes) in the presence or absence of PEG-catalase pretreatment; n=5. *P<0.05 as indicated. H, Representative Western blots and averaged data of p-PKC-δY311 and total PKC-δ from perfused rat hearts with or without IPC (10 minutes after the last reperfusion); n=4. *P<0.05 vs control group. I, Representative Western blots and averaged data of MG53 from the culture medium of NRVMs infected with Ad-β-gal, Ad-PKC-δ or Ad-PKC-δ-Y311F mutant with or without H2O2 treatment (100 µmol/L for 20 minutes); n=5. *P<0.05 as indicated. For A through I, data are presented as mean±SEM. For B, C, E, F, and I, data are normalized to the corresponding nonspecific bands (NS), which were obtained through brilliant green staining.
Because phosphorylation of PKC-δ at Y311, a highly conserved residue among species (Figure V in the Data Supplement), is involved in ROS-dependent PKC-δ activation,42,43 we next examined whether IPC-triggered MG53 secretion is related to ROS-induced phosphorylation of PKC-δ Y311. In cultured NRVMs, phosphorylation of PKC-δ Y311 was elevated in response to H2O2 treatment, which was attenuated by pretreatment of cells with PEG-catalase (Figure 4G). More important, IPC was able to increase PKC-δ Y311 phosphorylation in rat hearts, implying a crucial role of PKC-δ Y311 phosphorylation in IPC-induced MG53 secretion (Figure 4H). This possibility was further confirmed by the fact that enforced expression of the PKC-δ-Y311F mutant failed to enhance H2O2-induced MG53 secretion (Figure 4I). Furthermore, treatment of cells with either PP1 or bosutinib, Src kinase inhibitors,42 fully blocks H2O2-induced PKC-δ Y311 phosphorylation in NRVMs (Figure VI in the Data Supplement). As for the intracellular distribution of phosphorylated PKC-δ Y311, we found that under basal conditions phosphorylated PKC-δ Y311 is localized mainly in the cytosol but translocated to the plasma membrane and mitochondria compartment in response to H2O2 treatment in NRVMs (Figure VII in the Data Supplement), consistent with the previous notion.44 Taken together, our in vitro and in vivo data indicate that IPC triggers MG53 secretion by H2O2-evoked, Src-mediated PKC-δ phosphorylation at Y311.
Cardiac I/R Injury Is Alleviated by Systemic Administration of Recombinant MG53 Protein in Rats
Next, we aimed to investigate the role of the secreted MG53 from cardiomyocytes. Because it is well established that IPC is the most powerful intrinsic protective mechanism against cardiac I/R injury6 and that intracellular MG53 protects the heart through both membrane repair45 and activation of prosurvival pathways,16,17 we hypothesized that secreted MG53 may exert a cardioprotective effect and act as an effector of IPC protection. To test our hypothesis, we evaluated the potential cardiac protective effect of systemic delivery of rhMG53 (Figure VIII in the Data Supplement) in male rats subjected to I/R injury (Figure 5A). Treatment of rhMG53 overtly decreased the infarct size (infarct size/area at risk ) induced by I/R compared with the BSA-treated control group (11.9±1.8% versus 27.3±2.0%; P<0.01) without altering the area at risk/left ventricle ratio (Figure 5B and 5C). The in vivo administration of rhMG53 reduced the mortality from 44.7% (17 of 38 animals) to 5.3% (1 of 19 animals) in rats, suggesting that elevation of blood MG53 concentration can markedly ameliorate I/R-induced cardiac injury and animal death (Figure 5D). Cardioprotection by rhMG53 was also evidenced by decreased blood LDH concentration, reduced numbers of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive cardiac cells, and restored cardiac function in the rhMG53 group relative to the control (Figure 5E through 5G). Similarly, rhMG53 treatment effectively protected female rats from cardiac I/R injury (Figure IX in the Data Supplement).
Figure 5. Cardiac ischemia/reperfusion (I/R) injury is alleviated by systemic administration of recombinant MG53 (mitsugumin 53) protein in male rats.A, Schematic diagram showing the protocol for recombinant human MG53 protein (rhMG53) protection against cardiac I/R injury in vivo. B and C, Representative photos (B) and averaged data (C) of the infarct size and area at risk (AAR) of the rats subjected to cardiac I/R injury with BSA or rhMG53 protein treatment in vivo; n=10 for BSA, 13 for rhMG53. **P<0.01 as indicated. Scale bar is 2 mm. D throughF, Death rates (D), serum lactate dehydrogenase (LDH) concentration (n=17 for BSA, 13 for rhMG53; E), and cardiomyocyte apoptosis detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (n=5; F) of the rats subjected to cardiac I/R injury with BSA or rhMG53 protein treatment in vivo. G, Cardiac function accessed by echocardiography in male rats subjected to cardiac sham or I/R injury with BSA or recombinant MG53 protein treatment; n=8 for sham, 6 for BSA and rhMG53. **P<0.01 as indicated. For C through G, data are presented as mean±SEM. In D, the numbers above the bar indicate the number of the animals that died during ischemia and reperfusion/the number of the animals subjected to surgery. LV indicates left ventricle.
Extracellular MG53 Restores IPC Cardioprotection in mg53−/− Mice
Previous studies have shown that IPC cardioprotection is totally abolished in mg53−/− mouse hearts.16 Therefore, we sought to determine whether delivery of rhMG53 can restore IPC protection in the MG53 knockout mice. MG53 was detected in the hearts of mg53−/− mice 1 hour after intravenous injection of rhMG53 (Figure 6A and 6B), suggesting that extracellular delivery of MG53 protein can be deposited to the hearts. IPC did not exert any cardioprotective effects in mg53−/− mice treated with BSA, consistent with the previous reports.16 Although pretreatment with rhMG53 alleviated cardiac I/R injury in mg53−/− mice subjected to I/R, IPC combined with rhMG53 pretreatment further reduced I/R-induced myocardial infarction, cardiomyocyte death, and cardiac dysfunction (Figure 6C through 6E), indicating that the presence of extracellular MG53 is able to restore the cardioprotection of IPC in mg53−/− mice and that extracellular MG53, similar to its intracellular species, participates in IPC-mediated myocardial protection.
Figure 6. Extracellular MG53 (mitsugumin 53) restores ischemic preconditioning (IPC) in mg53−/− mice.A, Schematic diagram showing the protocol for recombinant human MG53 protein (rhMG53) restoration of IPC protection against cardiac ischemia/reperfusion (I/R) injury in vivo. B, Representative Western blot of MG53 from the hearts of mg53−/− mice with BSA or rhMG53 protein treatment. Purified rhMG53 was used as a positive control (PC). C, Representative photos and averaged data of infarct size and area at risk (AAR) of the mg53−/− mice subjected to cardiac I/R injury with or without IPC and with BSA or rhMG53 protein treatment in vivo; n=6 for BSA+I/R, rhMG53+I/R and BSA+IPC+I/R, n=7 for rhMG53+IPC+I/R. *P<0.05 as indicated. Scale bar is 1 mm. D, Representative photos and averaged data of cardiomyocyte apoptosis detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of the mg53−/− mice subjected to cardiac I/R injury with or without IPC and with BSA or rhMG53 protein treatment in vivo; n=5. **P<0.01 as indicated. Scale bar is 20 µm. E, Cardiac function accessed by echocardiography in mg53−/− mice subjected to cardiac sham or I/R injury with or without IPC and with BSA or rhMG53 protein treatment in vivo; n=6 for sham, BSA+I/R, and BSA+IPC+I/R; n=7 for rhMG53+I/R and rhMG53+IPC+I/R. *P<0.05 as indicated. For C through E, data are presented as mean±SEM. LV indicates left ventricle.
MG53 Is Present in Human Heart and Enables the Heart to Resist Oxidative Injury
Although previous studies have reported that there is little or no MG53 present in human heart,46 using a highly specific MG53 monoclonal antibody, we detected low expression levels of MG53 in human myocardial tissue (Figure 7A and Figure X in the Data Supplement). The identity of the band recognized by the MG53 antibody was verified by mass spectrum analysis, in which multiple peptides of MG53 were detected (Figure 7B). Regardless of its low expression levels in human heart, knockdown of MG53 with siRNA exaggerated H2O2-induced death of cardiomyocytes differentiated from human ES cells (Figure 7C and 7D), suggesting that MG53 may be required for the human heart to resist oxidative stress. Similar to NRVMs, the secretion of MG53 was induced in human ES cell–derived cardiomyocytes in response to short-term, low-dose H2O2 stimulation in the absence of a change in LDH concentration in the culture medium (Figure 7E and Figure XI in the Data Supplement). Last, rhMG53 treatment ameliorated H2O2-induced damage in human ES cell–derived cardiomyocytes (Figure 7F), further underscoring the potentially important biological function of MG53 in human heart.
Figure 7. MG53 (mitsugumin 53) is present in human heart and enables the heart to resist oxidative injury.A, Representative Western blots of lysates (50 µg) from the heart, liver, and skeletal muscle from normal human heart and the heart and skeletal muscle of wild-type (wt) or mg53−/− mice. Purified recombinant human MG53 protein (rhMG53; 20 ng) was used as a positive control. Nonspecific bands (NS), which were obtained through brilliant green staining, were used as loading controls. B, Human heart whole lysates were immunoprecipitated with the MG53 antibody and subjected to mass spectrum (MS) analysis. The peptides detected by MS experiments matched with MG53 sequence are shown in bold red. C, Representative Western blots and averaged data of MG53 from cultured human cardiomyocytes derived from human embryonic stem cells transfected with scrambled or 2 sets of MG53 siRNAs; n=4. *P<0.05 as indicated. D, Representative Western blots and averaged data of cleaved caspase-3 from the lysates of cultured human cardiomyocytes with H2O2 treatment transfected with scrambled or MG53 siRNAs; n=4. **P<0.01 as indicated. E, Representative Western blots and averaged data of MG53 from the culture medium of human cardiomyocytes with H2O2 treatment (at indicated dosages for 2 hours); n=4. *P<0.05, **P<0.01 as indicated. F, Representative Western blots and averaged data of Cleaved caspase-3 from the lysates of cultured human cardiomyocytes with H2O2 treatment (200 µmol/L for 20 hours) preincubation of BSA or rhMG53 protein (0.1 µg/mL for 3 hours); n=4. *P<0.05. Data are presented as mean±SEM. For E, data are normalized to the corresponding nonspecific bands, which were obtained through brilliant green staining.
Discussion
Although numerous cardiokines have been identified and characterized over the past 3 decades, here we have provided multiple lines of evidence to demonstrate that the most powerful intrinsic means of cardioprotection, IPC, elicits MG53 secretion via H2O2-evoked PKC-δ signaling, which, in turn, participates in IPC-mediated cardioprotection. First, we show that IPC-induced MG53 secretion is accompanied by increased H2O2 production and PKC-δ activation in rodents in vivo and in perfused isolated hearts. Second, H2O2 increases and its specific scavenger catalase blocks MG53 secretion and phosphorylation of PKC-δ at the Y311 site. Last, upregulation of PKC-δ enhances whereas its deficiency or the disruption of its Y311 phosphorylation prevents IPC- and H2O2-induced MG53 secretion. These results indicate that IPC-triggered MG53 secretion is attributable mainly to H2O2-activated PKC-δ signaling (Figure 8).
Figure 8. Schematic presentation showing that ischemic preconditioning (IPC) induces MG53 (mitsugumin 53) secretion through H2O2-protein kinase-C-δ (PKC-δ)–dependent pathway to mediate IPC cardioprotection. In response to repetitive short-term IPC, cardiac reactive oxidative species (H2O2) was increased, which induced the phosphorylation of Y311 site of PKC-δ and subsequently led to the secretion of intracellular MG53. The extracellular MG53, similar to its intracellular species, participates in cardiac IPC protection.
Extracellular MG53 has previously been explained by passive leakage caused by cardiac damage.46,47 However, this work and recent studies have revealed that MG53 can be secreted in response to physiological or pathological stress signals. For example, under metabolic stress conditions such as high glucose or high insulin stimulation, MG53 is secreted from the heart and skeletal muscle as a cardiokine/myokine, contributing to the pathogenesis of systemic insulin resistance.27 Here, we have illustrated a robust response of MG53 secretion to IPC or oxidative stress. Furthermore, we have elucidated that MG53 secretion is mediated by a canonical secretory pathway27 that requires the activation of H2O2–PKC-δ signaling in the absence of cardiac injury or cell membrane breakage. Functionally, the secreted MG53 participates in IPC-induced cardioprotective signaling and markedly ameliorates I/R injury. Thus, extracellular MG53, as a cardiokine/myokine rather than a passively leaked protein, regulates both cardiac function and myocyte viability in addition to its role in modulating insulin signaling and energy metabolism.
Several studies have reported that recombinant MG53 protein ameliorates various harmful stimulus-caused injuries to the heart, skeletal muscle, and some nonmuscle organs.47–54 Although intracellular MG53 protects the heart through the activation of reperfusion injury salvage kinase signaling,16 the mechanism underlying the protective effect of recombinant MG53 protein remained elusive until the present study. Here, we have found, for the first time, that, as an endocrine/paracrine organ, the heart secretes MG53 in response to IPC, which in turn participates in IPC-induced cardioprotection, as evidenced by the restoration of IPC protection through systemic delivery of rhMG53 in MG53-deficient mice (Figure 6). The ability of recombinant MG53 protein to rescue IPC cardioprotection in mg53−/− mice implies that exogenous MG53 may enhance or mimic IPC-mediated cardioprotection in human heart in which endogenous MG53 expression is limited. Taken together, these in vitro and in vivo studies not only shed new light on our fundamental understanding of the mechanism responsible for IPC cardioprotection but also underscore the high potential of using circulating MG53 as a novel therapeutic target to treat various human heart diseases.
It is noteworthy that either ROS or PKC-δ acts as a double-edged sword for the heart, although both are crucial for IPC-induced MG53 secretion and the resultant cardioprotection. Specifically, massive ROS production leads to cell injury and death, but a moderate short-term increase of intracellular ROS is an important mediator of cardiac preconditioning and indispensable for IPC-mediated cardioprotection.35,55,56 Mitochondria are the major source of H2O2 involved in preconditioning-triggered cardiac protection.15,57 Specifically mitochondrial respiration produces superoxide, a major component of ROS, which undergoes dismutation to generate H2O2. In line with the previous notion, we have shown here that IPC elevates H2O2 production, resulting in H2O2-dependent MG53 secretion and its participation in IPC protection. We have found that short exposure of cardiomyocytes to H2O2 is protective, whereas prolonged H2O2 treatment of cells is detrimental regardless of its concentration (50–200 μmol/L).
Similar to ROS, PKC-δ is essentially involved in cardiac IPC protection,39,58 but it also induces cardiomyocyte apoptosis under some circumstances.59,60 The different outcomes of PKC-δ activation may be related to the form and extent of its activation. For example, caspase cleavage of PKC-δ is an important mechanism for amplification of the apoptotic pathway61; however, phosphorylation of PKC-δ at Tyr155 is antiapoptotic in glioma cells.62 Here, our data show that IPC-induced, Src-mediated phosphorylation of PKC-δ at Y311 protects the heart against I/R injury through promoting MG53 secretion. Because PKC-δ Y311 is a target of Src42 and because both PKC-δ and Src are translocated to mitochondria in response to IPC or H2O2 treatment,44,63,64 we presume that PKC-δ is phosphorylated by Src on cardiac mitochondria and subsequently evokes MG53 secretion. Nevertheless, this possibility merits future investigation.
In addition to its extracellular versus intracellular distribution, posttranslational modification of MG53 plays an important role in its stability and biological function. Myocardial ischemia triggers MG53 S-nitrosylation at cysteine 144, which prevents oxidation-induced MG53 degradation, leading to cardioprotection.65 S-nitrosylation of MG53 and other proteins has previously been implicated in both cardiac IPC and postconditioning protection.66–68 In particular, MG53 cysteine 144 S-nitrosylation has been identified in postconditioning heart.67 Because the mutation of MG53 cysteine 144 prevents I/R-induced MG53 release from the heart,69 future investigation is required to determine whether S-nitrosylation of this site is obligated to IPC-mediated MG53 secretion.
Although it has previously been claimed that little or no MG53 is expressed in human heart,46 in the present study, Western blot, in conjunction with mass spectrum analysis, has revealed that MG53 is present in human left ventricle and its abundance is ≈1/10 of that of human skeletal muscle. As is the case in rodent cardiomyocytes, MG53 is secreted from human ES cell–derived cardiomyocytes in response to oxidative stress, and deficiency of MG53 sensitizes the cells to oxidative stress. Likewise, despite the fact that the amount of MG53 in murine kidney and lung is only 2.5% to 5% of that in skeletal muscle, its deficiency exaggerates tissue injury in mice.51,52 Thus, MG53 has biological functions in tissues with relatively low expression levels.
Conclusions
We have provided multiple lines of evidence to define MG53 as an IPC-sensitive cardioprotective cardiokine, marking circulating MG53 as a promising target for the treatment of cardiac I/R injury and other kinds of myocardial damage. Mechanistically, we have shown that MG53 secretion is triggered by IPC-induced increases in ROS, in particular H2O2, which subsequently activates PKC-δ via elevating its phosphorylation at Y311. Our present and previous findings indicate that circulating MG53, as a cardiokine, acts as a double-edged sword, protecting multiple organs from acute I/R injury and oxidative stress but, when chronically elevated, inducing insulin resistance and metabolic disorders. The present and previous studies pave the way for MG53 signaling pathway(s)-based therapies to treat ischemic heart diseases and cardiometabolic diseases.
Acknowledgments
The authors thank Drs Heping Cheng, Zhuan Zhou, and Claire Xi Zhang for insightful discussions and Haibao Shang, Wen Zheng, Lei Huang, Wenqian Zhang, Ning Hou, and Yu-Li Liu for their excellent technical support. Special thanks go to Dr Hiroshi Takeshima and Jian-jie Ma for their generous support in providing mg53−/− mice. The authors thank the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University and Dr Wen Zhou from the Analytic Instrumentation Center of Peking University for the analysis of mass spectrometry samples.
Sources of Funding
This work was supported by the National Key R&D Program of China (grant no. 2018YFA0800701, 2018YFA0507603, 2018YFA0800501), the National Natural Science Foundation of China (grant no. 31671177, 81630008, 81790621, and 31521062), the Beijing Municipal Science & Technology Commission (grant no. Z171100000417006), and the Beijing Natural Science Foundation (grant no. 5182010).
Disclosures
None.
Supplemental Materials
Expanded Methods
Data Supplement Figures I–XI
Data Supplement Table I
Footnotes
References
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