Introduction
Upon myocardial infarction (MI) immune system becomes activated by extensive necrosis of cardiomyocytes and release of damage-associated molecular patterns [
16]. Neutrophils are the first immune cells that infiltrate the damaged tissue as early as 24 h after injury. Then, 3–4 days later inflammatory monocytes are recruited into ischemic area of the heart. Apparently, overactive and prolonged immune responses can be responsible for heart failure in patients surviving the ischemic episode [
13,
21]. Thus, development of strategies targeting particular subsets of inflammatory cells and providing well-timed resolution of inflammation may be crucial for proper healing and recovery.
Heme oxygenase-1 (Hmox1) catalyzes the conversion of heme to biliverdin, carbon monoxide (CO), and ferrous iron (Fe
2+). Through the first two compounds Hmox1 mitigates cellular injury. This is accomplished by their antioxidant, anti-apoptotic, and anti-inflammatory effects [
7,
34]. We have previously demonstrated the impaired wound healing [
9,
20] and recovery after hind limb ischemia [
25] in Hmox1 knockout (Hmox1
−/−) mice when compared to their wild-type (WT, Hmox1
+/+) littermates. An important role of this protein was also demonstrated in different experimental studies on heart damage. Short-term cardiac stress, induced by ischemia–reperfusion (I/R) injury resulted in impaired left ventricular (LV) recovery and increased infarct size in heterozygous Hmox1-deficient (Hmox1
+/−) mice [
55]. In contrast, transgenic animals with cardiac-specific Hmox1 overexpression were better protected from I/R, with improved contractile recovery and reduced infarct size, inflammatory cell infiltration, oxidative damage, and apoptosis [
54]. Recently,
Hmox1 gene transfer was shown to attenuate post-ischemic inflammation in both murine and porcine I/R-injured hearts [
22]. In addition, beneficial effects of Hmox1 were demonstrated also under prolonged ischemia in mice with permanent ligation of the coronary artery [
47]. Myocyte-restricted Hmox1 transgenic mice exposed to MI exhibited significantly improved survival and LV function, lower interstitial fibrosis and oxidative stress. These beneficial effects were at least partially mediated by CO-dependent inhibition of mitochondrial permeability transition and apoptosis [
47].
Importantly, the findings in animal models are in accordance with several clinical investigations, since in humans
HMOX1 gene expression is modulated by a guanidine thymidine dinucleotide ([GT]n) repeat polymorphism in the promoter region (reviewed in: [
8]). Shorter repeats with (GT)n <25 are associated with higher inducibility and activity of HMOX1. On the other hand, the longer repeats result in lower HMOX1 expression and activity and were associated with an increased risk of cardiovascular disease (reviewed in: [
8]). Thus, heme oxygenase-1 is important for cardioprotection and repair, but its involvement in the suppression of post-ischemic inflammation remains incompletely understood.
Murine blood monocytes are generally divided into two main subpopulations. One is described as non-classical or non-inflammatory (Ly6C
+CD43
++; Ly6C
lo). These cells patrol the intravascular endothelial cell surface and clear dying endothelial cells [
37]. Another subset called classical or inflammatory (Ly6C
++CD43
+; Ly6C
hi) spikes during acute MI and accumulates in the evolving myocardial wound [
37]. Sometimes, also a third subset is distinguished—intermediate monocytes (Ly6C
++CD43
++; also Ly6C
hi), that are found at a lower frequency and to some extent resemble the classical monocytes, as they expand with cytokine treatment and in inflammation [
56]. In contrast to Ly6C
lo, the Ly6C
hi monocytes express on their surface a chemokine receptor CCR2 [
43]. Interaction between CCR2 and its ligand CCL2 (monocyte chemoattractant protein-1, Mcp-1), upregulated in the inflamed tissue, results in inflammatory monocyte recruitment [
10]. It was recently shown that the post-I/R influx of immune cells, including Ly6C
hi monocytes, was further exacerbated in Hmox1-deficient murine hearts [
22]. On the other hand, overexpression of
Hmox1 in the heart with gene therapy was able to revert this process [
22].
Importantly, under inflammatory conditions monocyte-derived macrophages gradually replenish the population of cardiac-resident macrophages [
15]. The latter ones are described as embryonically derived and capable of sustaining themselves by local proliferation [
15]. Importantly, their substitution with monocyte-derived macrophages may be responsible for decreased cardiac repair potential [
29]. Additionally, mechanical strain rising in the LV wall after MI, evokes macrophage proliferation [
41]. It was demonstrated that injection of nanoparticle-loaded siRNA targeting CCR2 to the atherosclerotic apolipoprotein knockout (ApoE
−/−) mice with MI reduces the recruitment of Ly6C
hi monocytes, improves the infarct healing and attenuates the post-MI heart failure [
35]. On the other hand, macrophages are important in resolution of inflammation. They produce anti-inflammatory cytokines and chemokines and eliminate tissue debris. In fact, either increased or insufficient macrophage expansion was demonstrated to impair tissue healing after cardiac injury [
14]. Evidently, Hmox1 is important for a proper maintenance and function of macrophages [
39,
49,
53].
At steady state, monocytes are produced in the bone marrow from hematopoietic precursors, but during inflammation also spleen is involved in generation, depot and deployment of these cells [
45]. The organ may host extramedullary hematopoiesis thanks to its ability to adopt hematopoietic stem and progenitor cells (HSPCs) liberated from the bone marrow in response to ischemic insult. Several reports demonstrated an important role of Hmox1 also in differentiation of lineage committed progenitors involved in erythropoiesis, thrombopoiesis, or B cell maturation (reviewed in: [
27]). However, involvement of this protein in monocytes generation and release from spleen following MI has not yet been investigated.
Thus, the aims of this study were to determine the role of Hmox1 in post-MI monocytes mobilization and their recruitment to injured cardiac muscle, as well as to check how different populations of cardiac macrophages are affected after MI in the presence and absence of Hmox1.
Materials and methods
Experimental animals
For our studies we used 12–14-week-old female Hmox1+/+ (WT) and Hmox1−/− specific pathogen-free mice of C57BL/6×FVB background. Age-matched mice were randomly allocated either to sham or MI surgery. All animal procedures were in accordance with Guide for the Care and Use of Laboratory Animals (Directive 2010/63/EU of European Parliament) and carried out under a license from the Ethical Committee of the Jagiellonian University. All animals were maintained under controlled environmental conditions (12 h light/dark cycle, at approx. 23 °C), and provided with standard laboratory food and water ad libitum.
Myocardial infarction surgery
Myocardial infarction was induced by permanent ligation of the left anterior descending (LAD) coronary artery as described previously [
36]. Mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (400 mg/kg of body weight). The limb withdrawal response to toe pinch was monitored to ensure the adequacy of anesthesia. Then, mice were placed on temperature-controlled, heated table, intubated and ventilated by connecting the 22G cannula inside the tracheal tube to a mechanical ventilator (MiniVent Type 854; respiration rate: 220 breaths/min, stroke volume 280 µl). The skin and muscles were cut along a breastbone, on the left side of a chest. Incision between 4th and 5th rib was made. Ribs were retracted with surgical threads and heart was exposed. Pericardial sac was opened, respiration rate was reduced to 150 breaths/min and LAD coronary artery was permanently ligated with 7.0 silk suture. Successful LAD occlusion was confirmed by an immediate color change of the myocardium supplied by the vessel from bright red to white. Initial respiration rate was restored and incisions between ribs, muscles and skin were closed with 6.0 silk suture. Mice were disconnected from the lung ventilator to allow normal breathing. In case of sham operated mice the procedure was the same, with placing the suture underneath LAD but without its ligation. Analgesia was applied to all (sham and MI-operated) animals twice daily for 3 consecutive days after surgery by subcutaneous injection of buprenorphine at the dose of 0.08 mg/kg of body weight.
Splenectomy
During isoflurane anesthesia the abdominal cavity of mice was opened above the left kidney. Then, the spleen vessels were carefully cauterized and spleen was removed. One month later mice were exposed to sham or MI surgeries as described above.
Detection of cardiac troponin I in plasma
Facial vein puncture was performed intravitally 24 h after each MI or sham surgery to obtain the peripheral blood (PB) anticoagulated with heparin (5 U/ml of blood). After centrifugation (800×g, 10 min) plasma was collected and stored frozen at −80 °C until further analysis. Cardiac troponin I (cTnI) was assessed in 100 μl of plasma with ELISA (DRG MedTek) according to vendor’s protocol.
Detection of hypoxic cells
To assess hypoxia of cardiac muscle Hmox1
+/+ and Hmox1
−/− mice were injected intraperitoneally with 60 mg/kg of body weight of pimonidazole hydrochloride (Hypoxyprobe, HP, Inc., MA, USA) and analyzed as previously described [
24,
26]. Thirty minutes after HP injection half of the animals in each group was subjected to LAD ligation. The other half was sham-operated. Then, 90 min after the surgery (and 2 h after HP injection) mice were killed for analysis of hypoxic cells with flow cytometry.
For preparing single cell suspensions from a heart, blood was washed out by injecting saline containing 0.5 U/ml heparin through the LV. Next, the whole heart was excised and both atria carefully cut off. Then, both ventricles were finely minced and incubated in 3 ml mixture of 5 mg/ml of collagenase type II (Gibco) and 1.2 U/ml of dispase (Gibco) in PBS with calcium and magnesium ions (Gibco) for 1 h at 37 °C with gentle agitation. Then, equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% Fetal Bovine Serum (FBS; Gibco) was added. Dissociated cells and undigested tissue were centrifuged at 200×g for 5 min and the pellet was resuspended in 5 ml of calcium and magnesium free PBS (Gibco). Cell/tissue suspensions were filtered through 100 µm cell strainers, centrifuged and resuspended in PBS containing 2% FBS. Then, the cells were stained for 30 min at 4 °C with 7-AAD (7-amino-actinomycin D) for the exclusion of nonviable cells. Next, the cells were washed with PBS, fixed and permeabilized for 20 min at room temperature (RT) with Cytofix/Cytoperm (BD Biosciences) according to vendor’s protocol. After washing, the cells were incubated with FITC-conjugated anti-HP antibody (Clone: 4.3.11.3, Hypoxyprobe, Inc., Massachusetts, USA) detecting protein adducts of pimonidazole in hypoxic tissue. The stained cells were analyzed using LSRFortessa cytometer (BD Biosciences) and Facs Diva (BD Biosciences) Software.
Transthoracic echocardiography
Mice were subjected to inhalation anesthesia with 2% isoflurane (Aerrane, Baxter) in air and immobilized on a heating platform ventral side up to maintain the body temperature at 37 ± 0.5 °C. Heart rate and respiration were continuously monitored by ECG electrodes. Mice chests were shaved and pre-warmed ultrasound gel was applied to the area of interest. Transthoracic echocardiography (TTE) was performed on day 7, 14 and 21 after MI using a Vevo 2100 system (Visual Sonics, Canada) with a 30-MHz transducer. The heart was first imaged in B-mode in the parasternal long axis (PSLA) view to examine the left ventricle (LV). Moreover, parasternal short axis (SAX) view was obtained at the level of papillary muscles. In case of each animal two-dimensional (2D) images crossing the anterior and posterior walls in PSLA and SAX were recorded. The following parameters were investigated: LV ejection fraction (LV EF, %), LV fractional shortening (LV FS, %), LV chamber volume (LV V, μl) and LV internal diameter (LV ID, mm) during both systole (s) and diastole (d).
Flow cytometry
At indicated time-points mice were anesthetized with 2% isoflurane (Aerrane, Baxter) in air and few drops of blood were collected by facial vein puncture to Microvette 100 EDTA tubes (Sarstedt) for white blood cell (WBC) count analysis using ABC Vet Hematology Analyzer (Horiba). Next, mice were euthanized by intraperitoneal injection of a mixture of ketamine (200 mg/kg of body weight) and xylazine (40 mg/kg of body weight). Peripheral blood was collected with a heparinized syringe from vena cava. Then, the animal was perfused with 5 ml saline containing 0.5 U/ml heparin through the LV. Heart, spleen, as well as femora and tibiae from both hind limbs were collected. Cells were isolated as described below.
Peripheral blood
Heparin blood samples (approx. 500–700 µl) were centrifuged (4 °C, 800×g, 10 min) and plasma was collected and stored frozen at −80 °C until further analysis. Then, all morphotic elements were suspended in 15 ml of hypotonic 1× RBC lysis buffer (formulation of working solution: 150 mM NH4Cl, 10 mM NaHCO3, 1 mM disodium EDTA, pH 7.4) for red blood cell (RBC) lysis and then centrifuged. If necessary, the lysis was repeated. After complete RBC lysis and removal of supernatant, cells were washed with PBS.
Spleen
The organ was cut in large pieces and pressed against a 100 µm cell strainer. Cells were washed with PBS and centrifuged. Then, the supernatant was discarded. Red blood cells were removed as described above. After complete RBC lysis and removal of supernatant, cells were washed with PBS.
Heart
Single cell suspensions from both ventricles of the heart were prepared as described in “
Detection of hypoxic cells”. Red blood cells were removed as described above. After complete RBC lysis and removal of supernatant, cells were washed with PBS.
Bone marrow
Epiphyses of femora and tibiae were cut and bone marrow was flushed from bones with PBS using a syringe with 30 gauge needle. Next, cells were centrifuged and red blood cells were removed as described above. After complete RBC lysis and removal of supernatant, cells were washed with PBS.
Before staining cells were incubated for 10 min on ice in PBS containing 2% FBS and anti-mouse CD16/CD32 antibody (clone 93, eBioscience) in concentration recommended by the vendor. Finally, cells were mixed with adequate panel of antibodies (Suppl. Table 1 and Suppl. Table 2) and stained for 25 min at 4 °C. For monocyte and macrophage staining, cell suspensions were incubated with antibodies directed against CD45, Ly6G, Ly6C, NK1.1, CD11b, CD11c, CD43, MHC II. For HSPCs, cell suspensions were incubated with antibodies directed against lineage markers (Ter119, CD3, B220, CD11b, Gr-1), Sca-1, CD117 (c-kit), CD34, CD48, CD150 (SLAM). In all staining procedures live cells were analyzed and dead cells were excluded by DAPI staining. Gating strategies for blood and spleen monocytes (Suppl. Figure 1), cardiac monocytes and macrophages (Suppl. Figure 2) and HSPCs in bone marrow and spleen (Suppl. Figure 3) are enclosed to Supplemental Material. The stained cells were analyzed using LSRFortessa cytometer (BD Biosciences) and Facs Diva (BD Biosciences) Software.
Western blotting
Pieces of tissue from infarct area and corresponding heart tissue from sham-operated mice (n = 2 mice/group) were collected on day 4, snap-frozen in dry ice and stored at −80 °C. Total protein was isolated by lysis of tissue in 300 µl of lysis buffer (1% Triton X-100 in PBS with protease inhibitors). Homogenization was performed using Tissue Lyzer (Qiagen). Protein concentration was determined using bicinchonic acid assay (BCA). For collagen type I detection, samples containing 40 µg of protein were prepared in non-reducing conditions and resolved in 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (wet transfer, 80 V, 2 h). For α-tubulin detection, samples were prepared in reducing conditions and subjected to the same procedure as described above. Membranes were blocked with 5% bovine serum albumin (BSA) in PBS containing 0.05% Tween-20 (collagen type I) or 5% milk in PBS containing 0.05% Tween-20 (α-tubulin) for 2 h at room temperature. Then, primary antibodies were added in 1:500 dilution (collagen type I, Abcam, ab21286) or 1:1000 dilution (α-tubulin, Calbiochem, DM1A) in blocking buffer and membranes were incubated overnight at 4 °C. On the following day, membranes were washed 5 times for 5 min with PBS containing 0.1% Tween-20. In the next step, secondary antibodies conjugated with horseradish peroxidase were added in 1:10,000 dilution in blocking buffer for 45 min at room temperature. After washing steps, chemiluminescent substrate for HRP activity (Immobilon Western Chemiluminescent HRP substrate, Merck Millipore) was added for 5 min and membranes were developed on photographic membranes.
Samples collection and histological examination
Histological analyses were performed in frozen sections of the hearts collected from mice 21 days after MI (n = 3 mice/group). The chest of anesthetized mice was opened and the heart arrested in diastole by intraventricular (LV) injection of 30 mM KCl in saline containing 0.5 U/ml of heparin. The right atrium was then cut and the heart was perfused with 5 ml of saline supplemented with 0.5 U/ml of heparin at a pressure similar to the mean arterial pressure via PE-50 catheter connected to a perfusion apparatus. After excision, heart was sliced into two transversal sections at the site of LAD ligation. The part below LAD ligation was embedded in OCT compound (Tissue-Tek) and snap-frozen in dry ice.
Assessment of cardiomyocyte hypertrophy
Sections were blocked with 1% BSA in PBS (1 h, RT) and then incubated with rhodamine-labeled wheat germ agglutinin (WGA; Vector Laboratories; dilution: 1:100, 30 min, RT). Sections were mounted with DAPI (4′,6-diamidino-2-phenylindole) containing medium to visualize nuclei. Analysis was performed at a 200× magnification using the ImageJ software by an observer blinded to the experimental protocol. For each sample, cardiomyocyte cross sectional area (CSA) was measured in 100 cardiomyocytes in which the nucleus was centrally located within the cell, in both the border peri-infarct zone and in the remote zone. The average regional cross-sectional area in μm2 were calculated.
Assessment of CD11b+ myeloid cells
Sections were blocked with PBS solution containing 10% goat serum, 1% BSA and 0.1% Triton (1 h 30 min, RT). After blocking tissue sections were incubated with primary rat anti-mouse CD11b antibody (BioRad, clone: 5C6; dilution: 1:200, overnight 4 °C). After washing in PBS the secondary anti-rat antibody (Alexa Fluor 488) was applied (dilution: 1:400, 1 h 30 min, RT). Sections were mounted with DAPI-containing medium to visualize nuclei. The cells were visualized in the peri-infarct zone at a 200× magnification.
Total RNA isolation, reverse transcription and quantitative PCR
Pieces of tissue from infarct area of the hearts and corresponding tissue samples from sham-operated and untreated mice were collected, snap-frozen in dry ice and stored at −80 °C. Total RNA isolation by lysis in 1 ml of Qiazol (Qiagen) per tissue sample was performed using Tissue Lyzer (Qiagen) followed by chloroform extraction and isopropanol precipitation. Concentration and quality of RNA was determined by 260 and 280 nm absorbance measurements using NanoDrop Spectrophotometer (Thermo Fischer Scientific). For cDNA synthesis 1 μg of RNA was used. Using NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen) cDNA was synthesized in total volume of 15 μl, according to vendor’s protocol. Obtained template for quantitative PCR (qPCR) was ten times diluted in ultrapure water. Quantitative PCR (qPCR) was performed using StepOne Plus Real-Time PCR (Applied Biosystems) with addition of SYBR Green PCR Master Mix (SYBR® Green JumpStart™ Taq, Sigma), 2 μl of diluted template and specific primers (Suppl. Table 3). Elongation factor 2 (Ef2) served as a housekeeping gene in mRNA analyses. In test samples, quantification of gene expression was calculated based on the comparative CT (threshold cycle value) method (ΔCT = CT gene of interest − CT housekeeping gene). The melting curve analysis was performed immediately after each qPCR for the presence of primer dimers or non-specific products.
Statistical analyses
Statistical analyses were carried out using GraphPad Prism software version 5 (GraphPad Software, Inc.). Results are expressed as mean ± SEM unless otherwise stated. First, values were tested for Gaussian distribution (Kolmogorov–Smirnov test). For two-group comparisons, an unpaired t test was applied to normally distributed variables, a Mann–Whitney test to non-normally distributed variables. For comparing more than two groups, a one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons, was applied. Survival curves were analyzed by Log-rank test. p < 0.05 was considered statistically significant.
Discussion
Following cardiac ischemia, the immune system has an important and complex role in driving the healing of injured myocardium. Better understanding of these multi-faceted cellular and molecular interactions may help to prevent the unwanted inflammatory damage, adverse cardiac remodeling and heart failure after ischemic episode.
Here we report that Hmox1 has a dual role in MI. On one hand, its deficiency is associated with better overall survival in the first 3–5 days post-ischemia due to much lower incidence of LVFWR. On the other hand, within the next few days Hmox1−/− animals develop greater LV dysfunction than the surviving Hmox1+/+ mice. This phenomenon was associated with increased post-MI mobilization of inflammatory Ly6Chi monocytes. Their increased cardiac recruitment resulted in a greater abundance of inflammatory MHC-II++ Ly6C+ CD11c+ macrophages in the hearts of Hmox1-deficient mice. Importantly, removal of splenic reservoir of monocytes limited the adverse late post-ischemic LV remodeling in Hmox1−/− mice.
In this study we used two methods to confirm the efficacy of the MI surgery and comparable in both genotypes initial infarct size. Measurement of hypoxic cells with hypoxia marker (pimonidazole hydrochloride) enables post-mortem analysis as early as 90 min after ischemia [
24,
26] and may constitute a good alternative for histological methods determining the infarct size, i.e., triphenyltetrazolium chloride (TTC) staining [
2]. The use of hypoxyprobe showed comparable numbers of hypoxic cells in the hearts of WT and Hmox1-deficient mice shortly after LAD ligation. The other measurement, evaluation of cTnI in the peripheral blood, was even more useful as mice with improperly induced MI, what sometimes happens due to anatomical variability of the left coronary artery branches [
1], had cTnI at the level of sham-operated animals and thus were excluded from further investigation.
Previous studies demonstrated the cardioprotective action of Hmox1 in both short-term [
22,
54,
55] (I/R) and long-term [
47] (permanent LAD ligation) ischemic cardiac stress. This can be attributed to the antioxidant, anti-apoptotic, and anti-inflammatory effects of the end-products of heme catabolism (biliverdin/bilirubin and CO) [
7,
34]. Additionally, Hmox1 may also act independently of enzymatic function, as its truncated form is found in the nucleus where it can regulate transcription factors involved in the antioxidant response [
31]. In our study,
Hmox1 gene was strongly upregulated in the hearts of WT mice 1 day after MI. Then, the expression decreased with time reaching the baseline on day 21 post-surgery. In fact,
Hmox1 gene contains one of the most regulated promoters identified so far. In addition to its substrate heme, several other compounds can also upregulate
Hmox1 expression, just to mention reactive oxygen species, inflammatory cytokines or hypoxia in rodents [
33]. All of these above-mentioned appear and can induce
Hmox1 expression under ischemic stress.
In the present study, we observed much lower occurrence of LVFWR and better overall survival in the first 3–5 days after permanent LAD ligation in mice deprived of Hmox1. According to clinical data, cardiac rupture most often occurs in patients with a large ST elevation MI (STEMI) and over the past few decades its occurrence has been greatly reduced due to higher use of reperfusion procedures and pharmacotherapy [
17]. Thus, the observed difference is mostly related to our experimental model of MI associated with a prolonged ischemia, whereas in short-term cardiac stress following I/R injury such differences may be covert. In animals, the likelihood of rupture after MI depends on species, strain, gender, and age (reviewed in Ref. [
42]). Experimental studies identified several gene products influencing rates of cardiac rupture (reviewed in Ref. [
42]). Among these, decreased collagen content has been associated with infarct expansion and rupture [
5,
18,
19,
50]. Hmox1 was previously described as vulnerable atherosclerotic plaque defining agent [
6] and its overexpression with gene therapy decreased interstitial collagen in rat hearts exposed to I/R injury [
32]. Moreover, it was recently shown that Hmox1
−/− mice exhibit a higher than WT mice degree of collagen deposition in their atria [
52]. To the best of our knowledge, here we report for the first time that the lack of Hmox1 significantly lowers the risk of LVFWR after permanent LAD ligation. The protective effect on this early rapid and deadly LV remodeling was associated with greater than in WT mice collagen type I production in the damaged area of the heart.
Next, starting from 7th until 21st day post-MI with TTE we observed an opposite effect—significantly impaired LV function in Hmox1
−/− mice, greater than in WT individuals. WGA staining performed 21 days after MI revealed greater cardiomyocyte hypertrophy in the LV peri-infarct zone of Hmox1-deficient mice than in their WT littermates. Hmox1 was previously shown to modulate the late post-MI remodeling by attenuation of LV hypertrophy and interstitial fibrosis [
38,
47]. These beneficial effects of Hmox1, and particularly one of its reaction product—CO, are at least partially mediated by regulation of cell cycle of different cells residing the heart—vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells and cardiomyocytes [
28,
30,
32,
48]. Additionally, cardiac monocyte-derived macrophages may intensify the post-MI LV remodeling and heart failure due to excessive immune response [
29].
Increased number of classical Ly6C
hi monocytes circulating in the blood of Hmox1-deficient mice and their enhanced post-I/R cardiac influx was recently associated with increased infarct size and impaired LV wall motion [
22]. In the present study we also detected greater numbers of inflammatory classical and intermediate Ly6C
hi monocytes in the blood of Hmox1
−/− when compared to Hmox1
+/+ mice both at steady state and 21 days post-MI. In addition, this was associated with prolonged upregulation of cardiac
Mcp-
1 gene expression, greater induction of adhesion molecules and increased numbers of monocyte-derived macrophages in the hearts of Hmox1-deficient mice with MI. Particularly, we noticed expansion of MHC-II
+ Ly6C
++ macrophage population starting from 4th until 21st day post-MI. It is well established that Ly6C
hi cells are the predominant subtype during the first few days post-MI, after which time Ly6C
lo macrophages begin to predominate [
37]. Additionally, among MHC-II
+ Ly6C
++, MHC-II
+ Ly6C
+ and MHC-II
++ Ly6C
+ macrophages, the CD11c
+ subsets expanded. On day 21, there was a significant difference in the number of all analyzed CD11c
+ macrophage populations between WT and Hmox-1-deficient mice subjected to MI. Contrary to cardiac-resident macrophages, the monocyte-derived macrophages were shown to strongly express on their surface CD11c antigen [
14]. The CD11c
+ macrophages represent a classical proinflammatory phenotype capable of efficiently producing IL-1β, TNF-α and IFN-γ [
23]. Moreover, elevated levels of MHC II (and other co-stimulatory molecules) on the surface (MHC
++) make these cells highly professional antigen-presenting cells capable of promoting strong lymphocyte responses [
23]. Expansion of these proinflammatory macrophage subsets in the infarcted hearts of Hmox1-deficient mice indicates their involvement in the heart failure progression.
Hmox1 is a critical regulator of the balance between self-renewal and differentiation of HSPCs under environmental stress as its haplodeficiency (Hmox1
+/−) was associated with premature depletion of HSC reserve following repeated myelotoxic injuries [
4]. In the bone marrow of Hmox1-deficient mice at steady state we detected more numerous populations of all investigated HSPCs. Moreover, GMPs on day 4 and SKL, ST-HSC, MPP on day 21 post-MI were more abundant in the bone marrow of Hmox1
−/− than Hmox1
+/+ mice. This indicates a more active hematopoiesis in the bone marrow of Hmox1-deficient mice subjected to MI.
In addition to bone marrow [
12], spleen may contribute to the post-MI monocyte mobilization. This organ was described as a site for storage and rapid deployment of monocytes [
45]. In this way, injury-induced monocytopoiesis usually does not lead to serious disturbances in generation of lymphocytes, erythrocytes and platelets in the bone marrow. In the spleen of intact Hmox1-deprived animals we detected higher numbers of different monocyte subsets—especially classical and intermediate which were significantly increased. On day 21 post-MI the numbers of classical and intermediate monocytes in spleens of Hmox1
−/− mice were lower than in their WT littermates. At the same time after MI, the numbers of these two monocyte subsets were increased in the blood of Hmox1-deficient mice, what may be a consequence of their departure from the spleen.
It was previously shown that following MI, upstream progenitors are mobilized from the bone marrow niches and engrafted in the spleen [
11,
40]. Activation of such supportive hematopoiesis leads to generation of surplus circulating monocytes. Analysis of different populations of HSPCs in spleen revealed an increase of SKLs and, among them LT-HSCs, on 1st day following MI in spleens of mice of both genotypes. Then, their numbers markedly dropped on day 4 indicating their exploitation in extramedullary hematopoiesis in the spleen. What is more, significantly lower steady state numbers of GMPs in Hmox1
−/− than Hmox1
+/+ mice were found. All these observations support a hypothesis, that the process of generation and liberation of monocytes from the spleens of Hmox1
−/− mice after MI is so intense, that in a consequence it leads to a rapid exhaustion of splenic reserve of hematopoietic progenitors.
We finally demonstrated an important role of spleen in the post-MI mobilization of the proinflammatory Ly6C
hi monocytes and heart failure progression in splenectomized Hmox1
−/− mice. Splenectomy significantly decreased the numbers of intermediate Ly6C
hi monocytes and prevented the post-MI expansion of the two inflammatory Ly6C
hi (classical and intermediate) subsets in the blood of Hmox1-deficient mice. As a consequence, the post-MI numbers of proinflammatory monocyte-derived macrophages, including CD11c
+ subsets of all analyzed macrophage populations were decreased in the hearts of splenectomized Hmox1
−/− mice and their heart function improved significantly when compared to non-splenectomized MI-operated Hmox1-deficient individuals. In contrast, removal of spleen in Hmox1
+/+ mice resulted in excessive post-MI numbers of classical Ly6C
hi and non-classical Ly6C
lo monocytes, increased numbers of monocyte-derived cardiac macrophages (but not in the CD11c
+ subsets) and a tendency towards greater LV dysfunction when compared to non-splenectomized MI-operated WT mice. Such discrepancies between splenectomized Hmox1
+/+ and Hmox1
−/− mice may suggest that Ly6C
hi classical and intermediate monocytes may have different origin. Intermediate monocytes in Hmox-1-deficient mice seem to derive from the spleen, as their numbers are dramatically reduced following splenectomy. A potent increase in the numbers of classical monocytes in the blood of splenectomized WT mice after MI indicates their bone marrow origin. This can further implicate different function of these cells in the injured cardiac muscle. In fact, Dutta et al. described different mRNA profile in monocytes of splenic and bone marrow origin and shown similarities in inflammatory gene expression between monocytes isolated from the spleen and Ly6C
hi monocytes isolated from atherosclerotic plaque after MI [
11].
A limitation of this study is that the mechanism by which Hmox1 regulates the production of monocytes still remains to be elucidated. The monocyte-macrophage lineage commitment at steady-state and during inflammation undergoes a tight control intrinsically via several transcription factors and extrinsically via different cytokines/growth factors [
44]. Thus, it would be interesting to investigate some of these mechanisms in the future.
In conclusion, our findings demonstrate a dual role of Hmox1 in MI. On one hand Hmox1 deficiency improves early post-MI survival by decreasing the occurrence of LVFWR. Afterwards, however, the absence of Hmox1 is associated with adverse late LV remodeling and severe heart failure after MI. This was associated with an increased monocyte mobilization to the blood and exaggerated myocardial macrophage infiltration. We identified spleen as an important source of the surplus circulating inflammatory Ly6Chi monocytes contributing to the severe LV dysfunction after MI in Hmox1−/− mice. Our findings may indicate that any therapeutic strategies aimed at modulation of Hmox1 expression in the heart post-MI should be applied in a timely manner.