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. Author manuscript; available in PMC: 2014 Sep 27.
Published in final edited form as: Circ Res. 2013 Jul 8;113(8):10.1161/CIRCRESAHA.113.301198. doi: 10.1161/CIRCRESAHA.113.301198

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through MER Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Elaine Wan 2, Xin Yi Yeap 1, Shirley Dehn 1, Rachael Terry 1, Margaret Novak 1, Shuang Zhang 1, Shinichi Iwata 2, Xiaoqiang Han 1, Shunichi Homma 2, Konstantinos Drosatos 2, Jon Lomasney 1, David M Engman 1, Stephen D Miller 1, Douglas E Vaughan 1, John P Morrow 2, Raj Kishore 1, Edward B Thorp 1
PMCID: PMC3840464  NIHMSID: NIHMS514585  PMID: 23836795

Abstract

Rationale

Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. Following myocardial infarction (MI), phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes (CMs). The molecular mechanisms of efferocytosis of CMs and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function.

Objective

We set out to identify mechanisms of dying cardiomyocyte (CM) engulfment by phagocytes and to for the first time assess the causal significance of disrupting efferocytosis during MI.

Methods and Results

In contrast to other apoptotic cell receptors, macrophage MER tyrosine kinase (MER-TK) was necessary and sufficient for efferocytosis of CMs ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic CMs, independent of changes in non-CMs, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk-/- mice corrected systolic dysfunction. Interestingly, an inactivated form of MERTK, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of MERTK inactivation post MI.

Conclusions

These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Keywords: Myocardial infarction, inflammation, macrophage, efferocytosis

Introduction

Efferocytosis is the clearance of apoptotic cells by phagocytes and requires the recognition of apoptotic “eat-me” signals by phagocyte efferocytosis receptors1-3. Whereas efficient efferocytosis activates pro-resolving/anti-inflammatory pathways in the phagocyte4, defective efferocytosis leads to secondary post-apoptotic necrosis and expansion of tissue necrosis5. Previous studies have linked defective apoptotic cell clearance to diseases of chronic non-resolving inflammation such as atherosclerosis and lupus6-12. In contrast, the extent to which efferocytosis efficiency during acute resolving inflammation may affect long-lasting organ function is much less clear.

Heart failure (HF) after myocardial infarction (MI) is a significant cause of morbidity and mortality13, 14. Though pharmacological advances, including β-blockers and angiotensin-converting-enzyme (ACE) inhibitors15, have significantly reduced mortality, the residual risk of post MI-induced heart failure remains high and is increasing16, 17. This necessitates the development of novel and complementary approaches to preserve heart function. The extent of cellular necrosis and apoptosis in the acute inflammatory phase of MI is a critical determinant of the degree of adverse remodeling leading to HF18-20. Therefore, strategies that enhance efficient resolution of inflammation and prevent unnecessary further cell death may be useful in slowing the progression to HF.

Inflammation post MI is an important component of healing after tissue injury21. A diverse population of bone marrow and spleen-derived immune cells are recruited to the heart after ischemic injury and promote clearance and repair of damaged myocardium22, 23. The innate immune response has beneficial activity in the healing heart24-26, however maladaptive cardiac inflammation can also be detrimental27, 28. Recent studies have identified innate monocyte/macrophage (MΦ) subsets to be differentially responsible for phagocytic and repair functions in the heart22. Beyond the identification of these cellular subsets, the molecular pathways responsible for dead-cell clearance in cardiac tissue remain unknown. Professional phagocytes interact with the myocardium in numerous capacities, including proteolysis29, dying/dead cell clearance, angiogenesis, and scarring30. In particular, inefficient removal of dead cardiac tissue has been linked to progression of heart failure28,30.

Professional phagocytes express multiple efferocytosis receptors to efficiently clear dying cells and prevent the release of autoantigens31. In this study, we interestingly discover that the MΦ efferocytosis receptor MERTK, which has been characterized by our group and independent labs8, 32-35, is specifically required for the clearance of dying adult CMs. Furthermore, we test for the first time the extent to which molecular pathways required for efferocytosis contribute to heart repair. Mertk+/+ and Mertk-/- mice were subjected to experimental MI and assessed for apoptotic CM accumulation, in vivo efferocytosis, inflammation resolution, infarct size, ventricular remodeling, and systolic function.

Methods

Briefly, CD36-/-36, LRPfl/fl LysMcre37, and Mertk-/-38 bone marrow-derived mouse macrophages were co-cultivated with primary adult mouse CMs and efferocytosis assessed ex vivo. Parallel assays were performed in which Mertk was reconstituted into cells to test sufficiency. In vivo, Mertk+/+ and Mertk-/- mice and bone marrow chimeras were subjected to myocardial infarction after permanent occlusion of the left anterior descending artery. Subsequently, Mertk expression (mRNA and protein), apoptotic cell accumulation (TUNEL), in situ efferocytosis, infarct size (TTC and collagen staining), inflammatory parameters (chemokine and cytokine mRNA and MΦ accumulation), and ventricular remodeling and cardiac function (echocardiography) were measured. The identification of glycosylated solMER in heart extracts was corroborated by addition of an N-glycanase. Statistical Analysis. Results are presented as means +/- SEM. Differences between multiple groups were compared by analysis of variance as appropriate and as indicated (2-way ANOVA and Bonferroni post-test), and differences between 2 groups were compared by unpaired Student t test (indicated by #). Two way repeated measures ANOVA was used to evaluate the statistical significance of data acquired from the same animal over multiple time points. A p value of < 0.05 was considered to be significant as indicated by * or #. Stated “n” values are biological replicates. Survival distributions were estimated using the Kaplan-Meier method and compared by the log-rank test.

An expanded and detailed Materials & Methods section is available in supplemental Online Data.

Results

Dying cardiomyocytes are engulfed by macrophage (MΦ) phagocytes

Previous studies have examined the consequences of MΦ and cardiomyocyte (CM) co-cultivation39, however and to our knowledge, the study of CM engulfment by phagocytes ex vivo has not been reported. To examine how CMs are ingested by MΦs, we co-cultivated dying primary adult mouse CMs with bone marrow-derived MΦs. After rinsing away non-engulfed cells, we could find evidence that ingestion of fluorescent CM bodies, indicated by red inclusions in green-labeled MΦs, occurred as early as 20 minutes after incubation (Fig. 1A). When co-cultivated at equivalent phagocyte: apoptotic-target ratios, the typical percentage of MΦs positive for ingestion of CM bodies was an inefficient 20-25%, compared with 30-40% under equivalent phagocyte/target ratios of apoptotic Jurkat cells, which are often used for in vitro efferocytosis studies1. Parallel confocal micrographs indicated that our rinsing protocol removed bound and non-ingested CMs and that internalization was specifically measured with this protocol. Furthermore, pre-incubation of phagocytes with cytochalasin D, blocked efferocytosis by disrupting actin polymerization (Online Figure IA).

Figure 1. Macrophages phagocytose cardiomyocytes (CMs) and Mertk is specifically required for CM efferocytosis.

Figure 1

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(A) Adult mouse CMs were isolated, fluorescently labeled (red), and induced to apoptosis. Dying CM apoptotic bodies were overlaid onto primary mouse MΦs and percent efferocytosis enumerated in Mertk+/+ and Mertk-/- MΦs. First image is a magnification of a MΦ ingesting a CM apoptotic body. In parallel, apoptotic Jurkat cells were co-cultivated with MΦs at equivalent ratios for efferocytosis quantitation. (B) Engulfment of CMs was measured after co-cultivating dying CMs with MΦs from CD36-/-, LRP deficient, or MER-/- primary MΦs. (C) Quantitation of efferocytosis after transfection of Mertk into Mertk-deficient HEK cells. MT=empty pIRES2 vector. (D) Bar graph showing TNFα mRNA expression by qPCR in Mertk+/+ vs Mertk-/- MΦs in the absence or after phagocytosis of CMs. Each column is mean± SEM. # indicates unpaired t test, p < 0.05. Asterisk indicates p < 0.05 relative to control (2-way ANOVA followed by Bonferroni post hoc test).

Identification of MERTK as a receptor specifically required for efferocytosis of CMs

A number of efferocytosis receptors have been identified in MΦs, including CD36, LRP, and MER tyrosine kinase (MERTK)38, 40, 41. To examine the potential role of these surface integral membrane proteins during efferocytosis of CMs, we co-cultivated MΦs from Cd36-/-, LRP-deficient (LRPfl/fl, LysMCre), or Mertk-/- mice with primary adult CMs. Apoptosis was induced in CMs by subjecting cells to ischemia (hypoxia and serum deficiency). Results were compared to littermate controls (Cd36+/+, LRPfl/fl, and Mertk+/+, respectively). The data show that Cd36 and LRP deficiency did not significantly affect engulfment, however, CM-associated fluorescence was greatly reduced (>70%) in Mertk-/- relative to Mertk+/+ phagocytes (Figure 1B). Similar requirements for phagocyte Mertk were found during ingestion of the murine CM cell line HL-142 (Online Figure IB). To determine if Mertk is sufficient for the engulfment of dying CMs, we transfected Mertk DNA into HEK-293A cells, which do not express Mertk43. Indeed, relative to cells transfected with empty vector (pIRES2-EGFP), ectopic expression of Mertk conferred the capacity of HEK cells to engulf CMs (Figure 1C). Interestingly, CM co-cultivation with MΦs induced TNFα and this was increased in the absence of Mertk (Figure 1D). Thus, MΦ Mertk specifically is necessary and sufficient for efferocytosis of cardiac CMs and suppresses CM-induced inflammation.

Exploring the role of Mertk in the heart

We sought to determine the physiological relevance of our findings in the heart. We first examined cardiac geometry and function from Mertk+/+ and Mertk-/- mice. Baseline indices of these mice are listed in Online Table I. Briefly, no significant differences in body mass or cardiac mass were found. By echocardiography, cardiac dimensions and contractile performance between adult Mertk+/+ and Mertk-/- mice were also initially similar. Microscopic examination did not reveal significant changes in CM size, apoptosis (by TUNEL staining), nor number of immune cells in adult mice at baseline.

Identification of Mertk expression in infarcted myocardium

We determined the extent of Mertk expression in the unique hypoxic milieu of the post MI heart. Figure 2 outlines a temporal and spatial analysis of Mertk expression in mouse myocardial tissue after wounding. Injury was induced by permanent occlusion of the left anterior descending artery (LAD), as we have recently described44. Semi-quantitative RT-PCR showed that non-infarcted hearts had relatively low Mertk expression. In contrast, we discovered that coronary occlusion led to significant induction of Mertk mRNA at 7 days post MI (Figure 2A). A time course analysis by quantitative RT-PCR (qPCR) in infarcted left ventricle (LV) vs. remote right ventricle (RV) revealed increases in LV Mertk as early as day 3 post MI and peaking at day 7 (Figure 2B). By laser capture micro-dissection of myocardial tissue sections, increases in Mertk mRNA were focused within the inflammatory border zone of the infarct (Figure 2C). MERTK protein levels paralleled mRNA (Figure 2D) and as expected, MERTK immune-reactivity was co-localized with F4/80+ MΦs (Figure 2E). We performed a time course analysis of heart extracts by flow cytometry and detected cell surface MERTK predominantly on monocytic Ly6cLO cells, in contrast to Ly6cHI monocytes, interestingly suggesting a role for differentiated MΦs45 in complementing Ly6cHI monocyte-mediated myocardial clearance, as previously reported22 (Figure 2F).

Figure 2. Identification and kinetics of Mertk expression post MI in experimental mice.

Figure 2

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(A) Semiquantitative RT-PCR of Mertk in heart extracts of Mertk+/+ and Mertk-/- mice 7days post occlusion (MI) of the left coronary artery. The first 2 lanes are from non surgically treated hearts. (B) Bar graph shows real-time PCR of Mertk mRNA in Left Ventricle (LV) and Right Ventricle (RV) at indicated days post MI. C = non-infarcted hearts. 7S = 7 days after sham infarction. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). (C) Bar graph of qPCR after laser capture microdissection of Mertk mRNA in remote (R) and ischemic (I) zones. (D) Immunoblot of MERTK protein levels in LV vs RV at indicated days (d) after MI. Densitometric analysis is plotted to the right. (E) Images show immunohisotchemical analysis of MERTK expression border zones of infarct. (F) Flow cytometric analysis was performed for Ly6c and MERTK levels on CD45-HI, CD11b-HI, Ly-6G-LO cells from myocardial extracts at indicated days before (PRE) vs. after MI. * indicates p < 0.05 after 2-way ANOVA followed by Bonferroni post hoc test. # indicates p < 0.05 after unpaired t test relative to control.

To assess the causal role of Mertk during MI, Mertk+/+ and Mertk-/- C57BL/6 littermates were subjected to MI as above and examined up to 28 days post infarction. No significant differences in mortality (7.3% in Mertk+/+ vs 9.1% in Mertk-/-, p = 0.32) or cardiac rupture were measured between experimental groups at the time points examined. Post MI, increases in heart mass were as expected, and heart-to-tibia-length and heart-to-body weight and were not different between the two groups (Online Table I). Also, no differences in baseline blood leukocyte or monocyte subsets were discovered (Online Figure II).

Mertk deficiency does not affect initial myocardial monocyte recruitment, however, leads to markers of delayed inflammation resolution

To determine the effect of Mertk deficiency on inflammation post MI, we examined monocyte levels and inflammatory cytokines. Online Figure IIIA shows that loss of Mertk did not affect absolute levels of monocytes in the heart prior and after MI. Similarly, the ratio of Ly6cHI and Ly6cLO monocytes were not affected (Figure 3A). Post-infarction inflammatory cells are recruited by rapid and transient induction of chemokine mRNA46. Post MI, Ccl2 (Mcp-1) was induced in WT mice with timely repression as expected23. Mertk-/- mice induced similar levels of chemokine and cytokine mRNA at early time points, however exhibited increases in Tnf-α and Il-6 and reduced levels of Il-10 at later time points (Figure 3B), consistent with a defect in inflammation resolution.

Figure 3. Chemokine and cytokine mRNA and inflammatory cells in hearts from Mertk+/+ vs. Mertk-/- mice.

Figure 3

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(A) Flow cytometric analysis of CD45-HI, CD11b-HI, Ly-6G-LO, Ly6c and CD11c cells in myocardium at indicated days (D) after MI. Enumeration in bar graph indicates percent monocytes that are Ly6c-HI vs. Ly6c-LO prior (P) or after MI at indicated days. (B) Time course (days = d) of chemokine mRNA expression in control and Mertk-/- infarcts. Measured were CCL2, TNF-α, IL-6, and IL-10. Data were obtained from 6 mice per genotype per timepoint. * indicates p < 0.05 relative to +/+ control.

Increased accumulation of apoptotic CMs coincides with a reduced index of efferocytosis in hearts of Mertk-/- mice after MI

Numerous reports have linked CM apoptosis to post MI heart repair, however, studies of CM fate after cell death are comparatively lacking. To first determine if Mertk is required for the accumulation of apoptotic CMs in vivo, we measured levels of CM (Desmin+) apoptosis (TUNEL+) at days 3, 5, and 7 post infarction in Mertk+/+ and Mertk -/- mice. CM apoptosis (Figure 4A) was increased (Figure 4A bar graphs) as expected after MI. However, no significant difference between experimental groups was found at day 3 post MI, consistent with a lack of effect of Mertk on initial infarct size (see Figure 5 below). This was not the case at later time points, which coincided with maximal expression of MERTK in WT hearts, as Mertk-/- hearts at days 5 and 7 exhibited significantly increased TUNEL+ CMs relative to control. To explore the possibility that elevations in apoptosis might be through enhanced susceptibilities to apoptosis, we isolated adult mouse CMs and subjected cells to ischemic stress. Importantly, CMs from Mertk+/+ and Mertk-/- mice exhibited similar kinetics of staurosporine and ischemia-induced cell death (Online Figure IV). Requirements for MERTK in vivo were not generalizable to accumulation of neutrophils (Online Figure IIIB), consistent with selective MERTK expression on Ly6cLO phagocytes, which emerge after reductions in myocardial neutrophil levels. Furthermore, Mertk deficiency did not affect the levels of TUNEL+ CD68 MΦs or TUNEL+ smooth muscle actin myofibroblasts, at 7 days post MI (Online Figure V). To directly assess if accumulations of apoptotic CMs were associated with defects in phagocytosis processing in vivo, we adapted a previously established methodology reported by our group and independent labs32, 47-50, and quantified tissue efferocytosis in situ. Similar to our ex vivo analyses, we enumerated the percentage of CM-associated MΦs by co-staining myocardial sections for CM Desmin and MΦ CD68. Figure 4B shows yellow CM Desmin signal co-localized with CD68+ phagocytes in border zones of the infarct. The quantified results of our blinded analysis are shown to the right. Two independent investigators achieved >91% concurrence after 15 hearts per group. In areas of equivalent phagocyte density, the measured percent Desmin-associated CD68+ cells was reproducibly and significantly reduced in Mertk-/- mice at day 5 post MI. Similar findings were found with the alternative CM marker α-actinin. These data are consistent with a defect in efferocytosis contributing to apoptotic CM accumulation.

Figure 4. Quantitation of cardiomyocyte (CM) apoptosis and association with CD68+ phagocytes in Mertk+/+ and Mertk-/- hearts post MI.

Figure 4

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(A) Photomicrographs of TUNEL nuclear (HOechst) staining in CMs (DESmin+). The arrows point to TUNEL (red) positive CM (green) nuclei (blue). TUNEL analysis is in the infarct border zone at indicated days (d) post MI. Bar = 200 micrometers. At 3days, bar graphs are quantitation of TUNEL positive CMs post MI relative to sham operated mice. At 5 days post MI, analysis is of Remote (R) and Border Zone (BZ) myocardium. 7day post MI analysis is within the BZ. (B) In situ efferocytosis analysis at 5 days post MI. Red phagocytes are CD68+. Phagocytes that also stain for CM Desmin are scored as efferocytosis as described in text. Bar = 100 micrometers. Similar findings were found with the alternative CM α-actinin. Asterisk = p < 0.05 relative to +/+ control.

Figure 5. Acute myocardial infarct size is increased in Mertk-/- mice post MI.

Figure 5

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Evan's Blue and TTC staining was performed in hearts at 3 (A) and 7 (B) days (d) post MI in Mertk+/+ and Mertk-/- mice as described in the Materials and Methods. AAR is Area At Risk and INF is Infarct. LV is left ventricle. * indicates p < 0.05 relative to +/+ control.

Deficiency of Mertk leads to increased infarct size

The extent of myocardial apoptosis is associated with the degree of myocardial necrosis, or infarct size, and infarct size is a major determinant of patient prognosis51. Thus, we were curious if deficiencies in apoptotic CM clearance might contribute to larger infarcts. To quantify infarct areas in hearts of Mertk+/+ and Mertk-/- mice, we performed TTC staining to differentiate live vs. dead tissue post MI and as we have previously described44. Importantly and for all time points assessed, the areas of endangered/ischemic myocardium (i.e., Area At Risk), as assessed by dye perfusion, were similar between genotypes, indicating equal and consistent levels of surgically-induced ischemia. The data show that, similar to levels of apoptotic cells (Figure 3), LV infarct size at day 3 post MI were not statistically different between Mertk+/+ (31% +/- 3%) and Mertk-/- mice (30% +/- 4%; p = 0.612) (Figure 5A). We hypothesized that if defective efferocytosis contributes to infarct area through secondary cellular necrosis, then accumulations in apoptotic CMs should precede changes in infarct size. Indeed, infarct size at day 7 (Figure 5B) was significantly enlarged with Mertk deficiency. Additionally, serum CPK (creatine kinase), an enzyme contained in viable myocytes and released into the bloodstream during myocardial injury, was also increased in the absence of Mertk (data not shown). These data are consistent with the hypothesis that non-cleared apoptotic CMs, conferred by defective Mertk, contributes at least in part to further loss of CMs and increase of infarct necrosis in injured hearts.

Mertk deficiency promotes adverse ventricular remodeling and LV functional deterioration post MI

Morphometric analysis of Mertk-/- mice 28 days post MI corroborated our findings of increased infarct size at day 7. Figure 6 shows that LV collagen area, as determined by Masson trichrome staining, was significantly increased in Mertk-/- mice compared to WT (10.38+/-0.78 versus 16.68+/-1.32; P<0.05). In addition, deficiency of Mertk led to thinner LV walls. Interestingly, picrosirius red staining under polarized light revealed that the degree of collagen cross-linking was also reduced in Mertk-/- mice (data not shown).

Figure 6. Quantitation of scar formation in remodeled hearts post myocardial infarction (MI) in Mertk deficient mice.

Figure 6

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(A) Representative photomicrographs of transverse cross-sections of trichrome-stained hearts from of control and Mertk-/- hearts at 28 days post MI. Picrosirus red staining was also performed and examined under brightfield and polarized light. (B) Quantitation of collagen levels after trichrome staining and PSR staining (polarized light) in the remote (R) and infarct (I) areas compared to non-infarcted control (c). (C) LV wall dimensions in Mertk+/+ and Mertk-/- hearts. * indicates p < 0.05 relative to +/+ control.

We asked if Mertk might contribute to heart performance post MI. As assessed by 2-dimensional M-mode echocardiography, infarction led to expansive LV remodeling and reduced systolic function in all mice as expected. Whereas LV volumes were similar between groups at 1 week post MI, Mertk-/- hearts exhibited enlarged LV end-diastolic volume (EDV) and end-systolic volume (ESV) by 28 days (Figure 7A). Increased LV remodeling was accompanied by further functional deterioration. Mertk-/- mice had significantly reduced systolic performance as indicated by depressed fractional shortening (FS) and ejection fraction (EF). Ventricular hemodynamic assessments also showed reductions in arterial systolic pressure and contractility in Mertk-/- mice after MI (Online Table I). To confirm that worsened heart contractility was the result of an infiltrating cell type and to evaluate the reproducibility of our findings, we generated Mertk-/- bone marrow chimeric animals after γ-irradiation as we previously described52. Protein and mRNA analysis of bone-marrow derived cells post engraftment validated successful depletion of myeloid Mertk in recipient animals (Online Figure VI). Similar to whole-body knockouts, deficiency of Mertk in bone marrow also led to suppressed EF and FS (Figure 7B, lower panel) after MI. In parallel, the reverse transplantation of Mertk+/+ bone marrow into Mertk-/- mice enhanced systolic performance relative to Mertk-/- receiving Mertk-/- marrow (p < 0.04).

Figure 7. Assessment of heart function by echocardiography after myocardial infarction (MI) in Mertk deficient mice.

Figure 7

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(A) Echocardiography analysis of volumes and % FS and % EF. ESV is end systolic volume. EDV is end diastolic volume. EF is fractional shortening. FS is fractional shortening. Images are M-mode tracings. (B) Assessment of FS and EF after bone-marrow transplant of either Mertk+/+ or Mertk-/- myeloid cells post MI relative to non-surgically treated control (c) mice. * indicates p < 0.05 relative to +/+ control.

Identification of low molecular weight MERTK products post MI

Our studies of targeted Mertk gene-deficiency led to a curiosity of natural mechanisms of MERTK regulation in the injured heart. Recent reports from our group and independent labs have characterized the inhibition of efferocytosis after proteolysis of an ectodomain fragment of MERTK, known as solMER43, 53. As shown in Figure 8, Western blot analysis identified significant MER immune-reactivity in a band consistent with the molecular weight of glycosylated solMER53; solMER was not detected in Mertk-/- mice. Furthermore, removal of N-linked sugars by glycanse treatment of myocardial extract led to collapse of this band to the precise molecular weight of the predicted 60kDa solMER ectodocmain. These data interestingly suggest a natural mechanism MERTK inactivation post MI.

Figure 8. Identification of a soluble MER profile post MI.

Figure 8

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Immunoblot of myocardial extracts 5 days post MI in WT mice. Three separate mice (MI1, MI2, and MI3) were subjected to ligation of the left anterior descending artery. Subsequently, myocardial extracts were interrogated for immunoreactivity against a monoclonal anti-MER (ectodomain specific) antibody. MI3 was also treated with a glycanse prior to electrophoresis.

Discussion

In light of decades of observations linking mobilization of the innate immune system to the repair of injured myocardium, the data herein provide strong support for the hypothesis that immune cell derived efferocytosis pathways bridge clearance of dying cardiac cells to subsequent repair and in turn, mitigate progression to heart failure. Through the utilization of a murine model of experimental MI, we report that Mertk deficiency in vivo leads to the accumulation of apoptotic CMs and a reduced index of in vivo efferocytosis. Suppressed efferocytosis preceded increases myocardial infarct size and led to subsequent delayed inflammation resolution. Importantly, Mertk deficiency led to compromised systolic performance. Ex vivo, Mertk was specifically necessary and sufficient for the efferocytosis of dying CMs. These findings suggest that, in addition to pro-inflammatory pathways that mobilize immune cells to the heart, pathways involved in resolution of inflammation30, 54, also play a key role in the healing heart. Furthermore, our findings are in line with previous reports identifying requirements for Mertk during the clearance of apoptotic cells in atherosclerosis9, 12, 55, thereby expanding the cardiovascular role of Mertk in a molecular continuum that connects pre- and post-MI events. This is interesting in that acute inflammation post MI is significantly lesser in duration compared to the chronic inflammation of atherosclerosis. We speculate that loss of precious non-regenerative CMs may underlie a heightened sensitivity to defective efferocytosis.

The findings of this study raise the important question of whether inefficiencies of efferocytosis occur naturally and are of significance in human clinical MI. Previous reports by Fragnogiannis and colleagues show defective necrotic CM clearance in aged experimental animals and an association with worsened heart repair28. However, this was also associated with a dampened immune response, preventing conclusions to be made regarding the intrinsic capacity of aged phagocytes for efferocytosis. During aging, it is interesting to note that phagocytes harvested from older animals exhibit reduced efficiencies of efferocytosis, the mechanisms of which are yet unclear48. In a similar light, Nahrendorf and Swirski have shown that hyperlipidemia is associated with delayed elimination of necrotic myocardium27. In this study, high cholesterol levels were associated with Ly6cHI monocytosis and an activated monocyte phenotype. Similarly, increases in circulating monocyte levels have been independently associated with poor patient prognosis after MI56.

If clearance of dying cells after MI is inherently inefficient or compromised in aged or hyperlipidemic patients, what mechanisms may be responsible? Though the goal of the current study was to deplete inflamed myocardium of a known efferocytosis receptor, it is also possible that MERTK could be rendered naturally dysfunctional in the setting of disease or genetic risk factors. For example, polymorphisms in Mertk are associated with increased autoimmune inflammation in diseases such as Lupus57, 58. In addition, ADAM metallopeptidase 17, which cleaves MERTK into a soluble inhibitory receptor43, 53, is increased during MI59. This may explain the identification of solMER in our murine extracts (Figure 8) and further provide the impetus for investigation of human MI specimens. MERTK activity may also be limited by availability of its ligand Gas6, which is required for binding to apoptotic cells60. Thus, it is tempting to speculate that studies herein mimic natural events and that limited evolutionary pressure during MI in the elderly may provide a window for therapeutic improvement.

It is important to consider other effects of Mertk within this study. For example, in some cases, Mertk deficient mice have a heightened inflammatory response38. In this study and previous reports32, the evidence does not support increased systemic or cardiovascular inflammation at baseline (Online TableI). In addition, initial inflammatory chemokine/cytokine levels, as well as initial recruitment of Ly6cHI cells, (Figure 3) was similar in all experimental groups. Furthermore, increased inflammatory levels and reduced IL-10 levels did not proceed accumulation of apoptotic CMs (Figure 4), all consistent with defects in the resolution arm of inflammation. Indeed, defective efferocytosis in general is associated with inflammation that is secondary to post-apoptotic cellular necrosis5. Thus, these findings argue for a direct role of Mertk within the ischemic myocardium. It will be important in future studies to examine the effects of defective efferocytosis or Mertk deficiency on other critical components of myocardial healing and remodeling. For example, elevated inflammation can further and directly affect ventricular remodeling by regulating myocyte phenotype transition, activation of matrix metalloproteinases, and TGF-β production30, 61. Regardless of the results of future studies on the mechanisms of MERTK in heart repair, the data reveal the importance of a single molecule in a critical stage of post MI heart repair, namely infarct size and reduced systolic function.

It is significant to mention that even in the absence of Mertk, accumulation of apoptotic cells eventually did subside and therefore other efferocytosis receptors and pathways almost certainly are involved in the heart. For example, thormobospondin-1 (TSP)-1, which prevents infarct-expansion through its effects on extracellular matrix62, is also required for apoptotic cell processing in vitro and therefore might also mediate its beneficial effects in the heart through efferocytosis63. Also of interest is the effect on efferocytosis by reperfusion after ischemia, the mechanisms of which may be distinct. Though a permanent occlusion model was utilized in this study to optimally examine the effects of Mertk on ventricular remodeling, future studies will examine the role of Mertk during reperfusion injury to explore therapeutic potential. Along these lines, the potential of harnessing clearance pathways in the heart has already been explored, as infusion of phosphatidylserine (PS) liposomes improves infarct repair through modulation of cardiac macrophage function64. PS is the canonical “eat me” signal that is recognized by phagocytes prior to efferocytosis.

The data herein identify bone-marrow derived Mertk as a significant determinant of cardiac repair and function after acute MI. From a broader perspective, these studies implicate efferocytosis as a causal modulator in the healing heart and merit the investigation of other myocardial efferocytosis pathways. Future studies are warranted in the setting of risk factors including aging and hyperlipidemia, as described above. Potential benefits of inflammation-resolution pathways post MI may also extend beyond heart failure and to secondary MI events downstream of atherosclerotic plaque rupture65, 66. Thus, these studies provide a proof of principle for the therapeutic potential of modulating the post MI inflammatory response through activation of pathways that link clearance of dying cells to inflammation resolution and heart repair.

Supplementary Material

1

Novelty and Significance.

What is Known?

  • Current increase in the prevalence of heart failure after myocardial infarction (MI) warrants novel therapeutic approaches.

  • MI leads to the recruitment of innate immune monocyte cells to the heart. These cells promote clearance and repair of dead myocardial tissue.

  • The molecular mechanisms and causal significance of phagocytic clearance of dying cardiac cells by monocytes is unknown.

What New Information Does This Article Contribute?

  • Monocytes/macrophages specifically require the cell receptor MER tyrosine kinase (MER-TK) for phagocytic uptake of dying cardiac cells.

  • Deficiency of MERTK from a monocyte subset with low levels of the marker Ly6c delays removal of cardiac cells and reduces systolic function post MI.

  • Clearance of dying cardiac cells may be limited by natural MERTK inactivation post MI.

This study was designed to understand the mechanisms and assess the causal significance of the molecular pathways that regulate the clearance of dying tissue after myocardial infarction (MI). The findings of the study suggest that MERTK, ae well characterized cell receptor, is selectively required for engulfment of dying cardiac cells by innate immune monocyte cells. We found that the loss of MERTK during MI suppressed removal of dying cardiac cells, delayed inflammation resolution and reduced systolic performance. Interestingly, MERTK was expressed on a select subset of previously characterized myocardial monocytes, indicating further specialization of monocyte function in the heart. In addition, we found evidence for natural inactivation of MERTK in the heart, suggesting that resolution of inflammation can be optimized post MI. These findings suggest that modulating the post MI inflammatory response by targeting molecular pathways that link clearance/phagocytosis of dying cells (efferocytosis) may be useful therapeutic strategy for promoting inflammation resolution and tissue repair after myocardial infarction.

Acknowledgments

Special thanks to Ira Tabas and Douglas Losordo for initital support of these studies. Thanks also to Prasanna Krishnamurthy, Alexander Mackie, and Sol Misener in FCVRI for surgical consultations. Thank you to William Muller and Hossein Ardehali for critical reading of the Manuscript. Thank you to Dr. Warren Tourtellote for histological collaboration.

Sources of Funding: This work was supported in part by an American Heart Association National Scientist Development Grant (# 09SDG2150036) and a NIH, National Heart, Lung, and Blood Institute grant 1K99/R00-HL09702 to ET.

Nonstandard Abbreviations and Acronyms

MER

myeloid-epithelial-reproductive

LAD

left anterior descending

HF

heart failure

LV

left ventricular

TTC

triphenyltetrazolium chloride

Footnotes

Disclosures: None.

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