Macrophages and the survival of cardiomyocytes after myocardial infarction

Abstract

Myocardial infarction (MI) causes severe burden to public health worldwide. After MI, stressed cardiomyocytes undergo necrosis and regulated cell death (RCD). Dynamic cell cascades ensue to limit tissue injury and promote healing. MI and subsequent ischemic injury trigger the influx of innate immune cells. Macrophages, among the first responders of MI, are known mostly to coordinate the scavenging of dead cardiomyocytes. However, rather than occurring as a homogenous population, macrophages have diverse ontogeny and pathological functions, exhibiting contradictory roles on the survival and RCDs of cardiomyocytes. This review focused on macrophages, and discussed their diverse dynamics and functional patterns after MI. We shared the insight of the roles of macrophages in cardiomyocyte survival and RCDs. We explored the use of single cell technology to discriminate specific macrophage clusters that can provide clues for precise targeting of cardiomyocyte survival after MI.

Graphical Abstract

Introduction

Mature mammalian hearts are notoriously non-regenerative due to the limited proliferative capacity of cardiomyocytes¹. Immediately after birth, mouse cardiomyocytes transit from glycolytic to oxidative metabolism². Increased oxygen consumption causes elevated reactive oxygen species (ROS), which force cardiomyocytes to exit the cell cycle². During MI, blockage of coronary vessels causes ischemic injury and acute cardiomyocyte death in cardiac muscle³. Timely reperfusion of the ischemic zone by Coronary Artery Bypass Graft or angioplasty remains the standard clinical treatment for MI⁴. However, abrupt reoxygenation during reperfusion can lead to ischemia reperfusion (I/R) injury, characterized by a second wave of cardiomyocyte death caused by inflammation, oxidative stress, and ion homeostasis disorder⁵. Ischemic cardiomyocytes were thought to only undergo necrosis and apoptosis, which cause acute and progressive loss of cardiomyocyte respectively⁶. However, recent studies using mouse models of MI indicate alternative RCDs in stressed cardiomyocytes that include ferroptosis, necroptosis and pyroptosis^7,8.

The occlusion of the left anterior descending coronary artery (LAD-O) in mice remains the most extensively characterized system in demonstrating the spatiotemporal and cellular dynamics of cardiomyocyte death and the innate immune response. Although not able to fully capture the complexity and heterogeneity of heart attack in human, it is widely used to mimic MI. A temporary occlusion of LAD captures the characteristics of I/R injury commonly observed in human patient after surgical intervention. Meanwhile, a permanent LAD-O results in a severe loss of cardiomyocytes and prolonged immune response as demonstrated in patients with long-term ischemic cardiomyopathy. Importantly, the permanent LAD-O is ideal for its efficiency to induce a high macrophage population, therefore subgroups with small population size can be detected^9,10.

The innate immune response is required to remove cellular debris and toxic metabolites from the infarct zone at early stage and to pave road for later wound healing stages. Phagocytes, such as neutrophils, peaks at day 1 and 2 after MI, followed by an influx of inflammatory monocytes marked by high level of lymphocyte antigen 6 complex C (Ly-6C). Inflammatory resolution is achieved by replenishment of reparative macrophages derived from both circulating and resident populations¹¹. Interrupting the innate immune response, especially in the early inflammatory stage, worsens the myocardial function and lower the survival rate^11,12.

Categories and spatiotemporal regulation of immune cells have been heavily reviewed^13,14. But their impact on cardiomyocyte death and survival was not often discussed. Macrophages are the most abundant immune population in injured hearts and are critical for the healing of the infarct. The macrophages act promptly in response to Damage-Associated Molecular Patterns (DAMPs) from cardiomyocytes, and paracrine factors from activated fibroblasts¹⁴. Later, macrophages downregulate proinflammatory genes, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF), while upregulate transforming growth factor-β (TGF-β) expression to foster wound healing¹⁵. During this transition, it is unclear whether the change of paracrine pattern in the microenvironment affect cardiomyocyte survival. The current review focused on the impact of macrophages on cardiomyocyte death during the biphasic immune response. We also expanded our perspective to the impact of signaling from dying cardiomyocytes on the function transition of macrophages.

1. Ontogeny and Functional Diversity of Cardiac Macrophages

The composition of the cardiac macrophage pool is not static. Macrophage heterogeneity and functions are dictated by both cell ontogeny and specific physical niches within the heart. C-C Motif Chemokine Receptor 2 (CCR2) expression and dependence distinguish cardiac macrophages of adult monocyte versus embryonic origin^16,17. CCR2⁻ cardiac resident macrophages originate from the yolk sac and take priority over antigen presentation and efferocytosis under ischemic conditions. In contrast, circulating monocyte-derived CCR2⁺ macrophages primarily coordinate cardiac inflammation¹⁸. Using high-resolution single-cell transcriptomics and multi-omics, a landmark study using human tissue and mouse models further identified three recurring, transcriptionally related macrophage subsets that exist across multiple murine organs, including the adult heart (Table 1)^19,20. These subsets are defined by unique core gene signatures and life cycles: a) The resident CCR2-TIMD4+ (T cell immunoglobulin and mucin domain-containing 4) subset, co-expressing distinct marker genes including lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Scavenger receptor cysteine-rich type 1 (CD163), and/or folate receptor beta (FOLR2), is highly conserved and maintained primarily through local self-renewal. This subset originates from both yolk sac and fetal monocyte precursors and is transcriptionally conserved across tissues and even species, suggesting a fundamental role; b) The CCR2⁺ subset relies on continuous replenishment from the circulating monocytes. It is crucial for mounting an inflammatory response after MI; c) The MHC-II^high subset, marked by high level of major histocompatibility complex class II (MHC-II), receives modest contribution from both monocyte influx and self-renewing (Figure 1).

Table 1:

Cardiac Macrophage Subsets: Origins, Markers, and Functions

Macrophage Subset	Developmental Origin	Key Surface Markers	Primary Function
Resident TIMD4+ (TIMD4+LYVE1+FOLR2+)	Yolk sac / Fetal monocytes	TIMD4, LYVE1, FOLR2, CX3CR1low, MHC-IIlow, CD163	Homeostasis, tissue repair, self-renewal, metabolic support30,31.
CCR2+ (TIMD4−LYVE1−FOLR2−)	Adult circulating monocytes	CCR2, Ly-6Chigh	Inflammatory response, recruitment, debris clearance19.
MHC-IIhi (TIMD4−LYVE1−FOLR2−CCR2−)	Mixed (Monocyte-derived)	MHC-IIhigh	Antigen presentation, modest monocyte contribution, not continuously replaced19.

Figure 1. A dynamic continuum of macrophages in the infarcted myocardium.

Within 24 hours After MI, monocytes derived from bone marrow and spleen are mobilized in response to the DAMPs, CCL2 and Alarmins from dying cardiomyocyte. They infiltrate the infarct zone, differentiate into macrophages, and join forces with self-renewing resident macrophages and MHC-II^hi macrophages to clear cell debris. Inflammatory chemokines from bone marrow-derived CCR2⁺ macrophages amplify the immune response, which is constrained by splenic CD169⁺TIMD4⁺ macrophages. At 7 days post-MI (DPMI), CCR2⁺ macrophages coordinate with splenic CD169⁺TIMD4⁺ macrophages for a transition to the resolving/healing phage.

Heterogeneity of macrophages is determined by the physiological status of their progenitors. Located at the apex of the hematopoietic system, the hematopoietic stem cells (HSCs) in bone marrow are a major source of circulating Ly-6C^high monocytes²¹. These cells tend to polarize toward a pro-inflammatory phenotype critical for clearing necrotic cells and initial healing. Stress such as aging can disturb the quiescence of HSCs and lead to clonal hematopoiesis (CH). CH is a process for certain HSCs with acquired mutations in a “driver” gene to gain competitive growth advantage, which causes clonal expansion of these mutant cells²². Ultimately, the percentage of circulating leukocytes with CH-driving mutations increases, leading to the formation of distinct clusters of blood cells such as monocytes²². An individual’s immune response can be affected by CH likely due to altered immune cell heterogeneity. Clinically, CH has been associated with MI, increased chronic inflammation in heart failure patients, and an increase in mortality caused by cardiovascular disease^22,23. Besides bone marrow, the splenic red pulp also serves as a reservoir of monocytes that can be mobilized at the early stage of MI, in response to the DAMPs and alarmins released by damaged myocardium^24,25. Notably, Ly6C^high monocytes in the blood and spleen exhibit essentially indistinguishable transcriptomic profiles, a similarity further validated at both mRNA and protein levels, indicating that splenic monocytes are functionally equivalent to their circulating counterparts prior to mobilization. Splenectomy reduces the influx of Ly6C^high monocytes into the ischemic myocardium by approximately 75%, indicating that the spleen mobilizes monocytes en masse in response to injury²⁴. Fate-mapping approaches using congenic transplantation models further demonstrate that a substantial fraction of monocytes accumulating in the infarcted heart are spleen-derived, thereby contributing to the heterogeneity of cardiac macrophage populations. Among these, the spleen-derived CD169⁺TIMD4⁺ macrophages are vital for efferocytosis in the infarct zone (Figure 1), promoting myocardial repair, suppressing inflammation, and limiting adverse remodeling²⁶. Meanwhile, bone marrow–derived hematopoietic progenitors are mobilized following MI and can migrate to the spleen, where they support extramedullary hematopoiesis, thereby sustaining monocyte production over time²⁷.

Functions of macrophages can also be determined by their spatial localization, a phenomenon termed niche-specific functional programming²⁸. For instance, Lyve1^lowMHCII^high macrophages preferentially locate near nerve bundles, and Lyve1^highMHCII^low macrophages reside alongside blood vessels²⁰. Depletion of the Lyve1^highMHCII^low macrophages exacerbates inflammation and collagen deposition, suggesting a critical role in restraining the inflammatory response. This phenomenon is further exemplified by the discovery of a specialized population of cardiac macrophages, which densely intersperse with conducting cells, express the gap junction protein connexin 43, and are shown to actively facilitate electrical conduction²⁹. This non-canonical role demonstrates how the tissue niche can direct the function of macrophages to fit into local physiology. The niche-dependent programming provides valuable insights into the heterogeneity of macrophages in infarcted myocardium.

2. The Macrophage after MI: A Dynamic Continuum

While aforementioned three major macrophage subsets are defined by ontogeny and steady-state function, these lineages form the hierarchical backbone upon which more transient, functionally diverse states emerge after MI. Multimodal single-cell analyses have revealed that macrophages rapidly transition through a spectrum of activation states, exhibiting elevated expression of genes involved in cytokine signaling, lipid metabolism, and tissue remodeling³². This dynamic transcriptional reprogramming reflects the adaptability of macrophages to local cues within the evolving infarct microenvironment. For example, the immediate aftermath is a short period (3 days) of sterile inflammation, triggered by cardiomyocyte death and the release of chemokines and DAMP³³. A transient but powerful burst of C-C chemokine ligand 2 (CCL2) draws Ly-6C^highCCR2⁺ monocytes from the bloodstream to the infarct zone (Figure 1). Once arrived, they differentiate into pro-inflammatory macrophages with strong expression of interferon pathway genes, e.g. interferon stimulated exonuclease gene 20 (Isg20), Isg15, interferon regulatory factor 7 (Irf7), interferon induced protein with tetratricopeptide repeats (Ifit), and interleukin 1 beta (Il1b)¹⁹. The primary function of these macrophages is efferocytosis, a process essential for dead cell clearance and maintaining homeostasis (Figure 1). However, the rapid accumulation of the pro-inflammatory macrophages also propagates inflammatory signaling. Interferon gamma (IFN-γ) from lymphoid cells and macrophages promotes the production of CCL2 to further recruit macrophages to the infarct zone^34,35. IFN-γ also stimulates the production of TNF-α in macrophages through IFNγR-mediated signaling³⁶. Myelomonocytic cells require IFN-γ signaling to synthesize and release IL-12, which in turn activates NK cells to release IFN-γ³⁷. The propagation of cytokine signaling ensure the activation of macrophages. Depletion of phagocytic or myelomonocytic cells cause abrogation of immune cell influx and deterioration of left ventricular function and survival after MI^11,12.

Concomitantly, the splenic CD169⁺TIMD4⁺ cells from the circulating Ly-6C^low monocyte population were also recruited to the heart immediately after MI²⁶. Once arrived, they differentiate into CD169⁺TIMD4⁺ macrophages (Figure 1). Compared to the resident TIMD4⁺ cardiac macrophages, the spleen-derived CD169⁺TIMD4⁺ subtype has a similar population size and lower level of LYVE1. Splenectomy in mouse MI models significantly deceases the cardiac CD169⁺TIMD4⁺LYVE1^low population and increases the mortality caused by cardiac rupture²⁶. The mortality can be rescued by intravenous infusion of donor splenic CD169⁺TIMD4⁺ cells, supporting their roles in post-MI acute inflammation and wound healing. Transcriptomic studies using mouse heart tissue collected during acute infarction period showed that the spleen-derived CD169⁺TIMD4⁺LYVE1^low macrophages express higher level of genes with functions in wound healing, extracellular matrix (ECM) remodeling and immunomodulation, compared to their LYVE1^high counterpart and bone marrow-derived clusters²⁶. By producing anti-inflammatory interleukins (IL-10, IL-4) and expressing suppressor of cytokine signaling-3 (SOCS3), these spleen-derived macrophages prevent excessive immune response and pave road for later resolution phase³⁸. Importantly, the spleen-derived CD169⁺TIMD4⁺ macrophages and cardiac resident macrophages form a population with a broad spectrum of anti-inflammatory function to restrain the acute immune response and ensure a timely and efficient phase transition (Figure 1).

The transition from the pro-inflammatory to a reparative phase is a hallmark of inflammatory resolution. The initial population of Ly-6C^high monocytes that first infiltrates the myocardium orchestrates both phases of the immune response and partly give rise to a second wave of reparative population. A key multifaceted macrophage population is the TREM2^high subset, which demonstrates similarities to lipid-associated macrophages and found in both human ischemic hearts and mouse MI model³⁹. They continue to perform efferocytosis, but also communicate with stromal cells, such as fibroblasts, to regulate extracellular matrix deposition and fibrosis^9,39. The formation of a stable, organized fibrotic scar is vital for maintaining the structural integrity of the infarcted ventricular wall and preventing catastrophic rupture³³. The outcome of this biphasic process hinges on a tightly regulated communication loop between immune and non-immune cells. Without proper spatiotemporal dynamics, the healing process becomes maladaptive.

3. Macrophages and Cardiac Regeneration

Cardiomyocytes are at the center of regenerative events after MI. Preserving the remaining cardiomyocytes and replacing dead cells with fibrosis are the main goals of the reparative response. Unlike amphibians and fish, adult mammalian hearts cannot fully regenerate after MI due to insufficient cardiomyocyte renewal⁴⁰. However, damaged neonatal mouse cardiomyocytes can reenter the cell cycle to populate the injured myocardium⁴⁰. Although this reparative ability is lost in juveniles, studies of the early regenerative window in comparison to the adult stage have identified multiple genetic pathways that can promote adult cardiomyocyte proliferation¹. These so-called healing factors includes Neuregulin-1 (Nrg1), an injury-induced cardiomyocyte mitogen⁴¹. Originally thought to be mainly expressed by endothelial cells, recent studies suggest macrophages of a significant source of Nrg1⁴². In a neonatal mouse MI model, the CCR2⁻ macrophages promotes cardiomyocyte proliferation, at least partially by secreting a cocktail of cytokines and growth factors including insulin-like growth factor 1 (Igf-1), oncostatin M (Osm), neuropilin 1 (Nrp1), phospholipid transfer protein (Pltp), alpha-L-fucosidase 1 (Fuca1), and Serpin family G member 1 (Serping1)^43,44. These healing factors are reported to be produced exclusively by cardiac resident macrophages, but recent studies suggested the spleen-derived CD169⁺TIMD4⁺ macrophages as another significant source of Nrp1²⁶. On the contrary, secretome including exosomes from inflammatory macrophages inhibit cell cycle activity in cardiomyocytes after MI⁴⁵. Although adult hearts have reparative macrophages after MI, it seems that their population size and/or activity is not sufficient for the cell cycle re-entry of cardiomyocytes. Their roles in cellular events other than the cardiomyocyte proliferation has not drawn enough attention. In the rest of the review, we expanded our focus to the impact of macrophages on cardiomyocyte death.

4. Macrophages and Cardiomyocyte Death after MI

4.1. Inflammatory death of cardiomyocytes:

cardiomyocyte death after MI has a significant impact on infarct size and cardiac remodeling. For decades, apoptosis and necrosis were thought to be the only ways of cell death, and were heavily studied in MI⁴⁶. Apoptosis limits inflammation which is required for cardiac repair after MI⁴⁶. Although apoptotic cardiomyocytes have been observed in animal models of MI, the low rate of apoptosis could not explain the scale of cardiomyocyte loss after MI⁴⁷. Recent studies from multiple groups assessed alternative RCDs and reported the involvement of ferroptosis, necroptosis, and pyroptosis in MI (Figure 2)⁸. In neonatal mouse heart, necroptosis occurs in cardiomyocytes within 6 hours after MI, and ferroptotic cardiomyocytes was observed throughout the biphasic inflammatory response⁷. These findings suggest that necroptosis causes the initial acute cardiomyocyte injury, and ferroptosis drives progressive loss of cardiomyocytes. Both types of RCDs were also reported in adult mouse model of MI⁸.

Figure 2. Interaction between macrophages and stressed cardiomyocytes.

In the infarcted myocardium after MI. CCR2⁻ resident macrophages and monocyte-derived CCR2⁺ macrophages interact with cardiomyocytes undergoing different types of RCDs including necroptosis, pyroptosis, and ferroptosis. Pro-inflammatory and pro-regenerative factors are produced and exchanged among these cells. Dying cardiomyocytes-derived IFN-γ and CCL2 attract circulating monocytes which differentiate into CCR2⁺ macrophages. The recruitment is propagated by autocrine of IFN-γ by the CCR2⁺ macrophages. They also interact with cardiomyocytes though TNF-α, IL-1β signaling and ROS to regulate cardiomyocyte survival. Meanwhile, cardiomyocyte-derived factors such as the IL-19 promote the population of reparative macrophages to control inflammation.

Considered more controllable than necrosis, the necroptosis is triggered by micro-environmental disturbance which activates toll-like receptors, and/or death receptors including Fas and TNFR1^48,49. Active receptors recruit receptor interacting serine/threonine kinase 1 (RIPK1). RIPK1 activates RIPK3⁵⁰, which then phosphorylates mixed lineage kinase domain like pseudokinase (MLKL). The activated MLKL homo-trimers can then disrupt cell membrane integrity^51,52. As a key necroptotic regulator, RIPK1 can activate NF-κB, resulting in production of cytokines and chemokines for the inflammatory phase⁵³. Meanwhile, ferroptosis is a non-apoptotic form of RCD caused by excessive production of lipid hydroperoxides in the presence of iron⁵⁴. The substrates for peroxidation are phospholipids with polyunsaturated acyl tails (PL-PUFAs)⁵⁵. By far, the most reliable morphological identity of a ferroptotic cell is shrunken mitochondria with increased membrane density and decreased mitochondrial cristae⁵⁶. Cellular iron, normally stored in a protein complex composed of ferritins, is required for the onset of ferroptosis⁷. ROS originate from the Fenton reaction and oxidize PL-PUFAs into lipid hydroperoxides, which trigger ferroptosis⁵⁶. Similar to necroptosis, ferroptosis is considered a form of regulated necrosis. Although they both result in leaky cell membrane and release of DAMP and cytokines, for necroptosis, the cell membrane rupture is an early and essential execution event, while the membrane rupture during ferroptosis occurs at a late stage as a consequence of extensive lipid damage. In addition, the serum levels of gasdermin D (GSDMD) and IL-18 were increased after angioplasty in patients with MI⁵⁷, suggesting that pyroptosis is also involved in cardiomyocyte loss after MI. Featured by rapid cell membrane rupture induced by GSDMD, pyroptosis is activated by NOD-like receptor 3 (NLRP3) inflammasome and more specialized for inflammation⁵⁸. Currently, it is not clear if these three types of RCDs are spatiotemporally separated after MI. While all show inflammatory features, evidence are needed to decide which one is more inflammatory or pro-tissue-repair.

Stressed cardiac cells have significant impact on inflammation and outcomes of MI (Figure 2). For example, ferroptotic cardiomyocytes and activated cardiac fibroblasts secrete IFN-γ and IL-8 respectively^7,59. Indeed, ferroptosis is crucial for triggering sterile inflammation after MI. With cell membrane damaged by lipid peroxidation, cytokines and DAMP are released to attract neutrophils and activate macrophages⁴⁶. CCL2 (MCP1), regarded as the most powerful chemokine to mobilize monocytes, is strongly released by ferroptotic cardiomyocytes in vitro⁵⁹. In neonatal mouse heart, ferroptotic cardiomyocytes produce IL-19 which drives the polarization of reparative macrophages, hence the pharmaceutical inhibition of ferroptosis did not improve heart repair⁵⁹. Likewise, necroptosis and pyroptosis jointly mediate progressive inflammation. After MI, the levels of RIPK1 and RIPK3 in the border zone are significantly upregulated for months⁸. NLRP3 inflammasome activation was observed in the infarct and border zone for an extended period until reparative phase⁸. In addition, GSDMD, the executor of pyroptosis, is released from pyroptotic cardiomyocytes and may act as a DAMP during the inflammatory process⁶⁰. These reports suggest that necroptosis and pyroptosis are persistently activated to induce progressive injury and adverse remodeling. Their key pathways seem to intertwine. It has been reported that the RIPK3/MLKL pathway is associated with inflammation through activation of the NLRP3 inflammasome and IL-1β in the absence of necroptosis⁶¹. The physiological function of inflammasome in cardiomyocytes needs further study. However, just like the biphasic inflammatory process, whether cardiomyocytes also have a multi-phasic death process to facilitate phagocytosis and fibrotic remodeling will need further investigation.

4.2. Detrimental Roles of Macrophages after MI:

The roles of macrophage in the RCDs of cardiomyocytes after MI have not been extensively studied. A dysregulated or prolonged inflammatory response, primarily driven by monocyte-derived macrophages, can create a pathological environment which compromises cardiomyocyte viability. A sustained inflammation can also impair communication with fibroblasts. Failure to resolve the initial inflammatory phase increases the risk of cardiomyocyte loss and ventricular rupture³³. Therefore, macrophages can at least indirectly impact cardiomyocyte survival. Meanwhile, emerging evidence implicate a direct role of macrophages in the regulation of cardiomyocyte death after MI (Figure 2). In early inflammatory phase, CCR2⁺ macrophages and neutrophils release pro-inflammatory cytokines such as TNF-α and IL-1β. TNF-α can bind to TNFR1 on stressed cardiomyocytes to induce necroptosis through the RIPK1/RIPK3/MLKL pathway. Alternatively, it can activate caspase-8 to induce apoptosis⁶². TNF-α also promotes mitochondrial dysfunction and ROS production in cardiomyocytes⁶³. The sheer number of macrophages in the infarct zone makes them a potentially significant source of ROS and reactive nitrogen species (RNS)⁵⁹, both can cause irreversible damage to adjacent cardiomyocytes. While ROS (superoxide and H₂O₂) posts threat on lipids, protein and DNA, the nitric oxide (NO) and peroxynitrite (ONOO⁻) from macrophages can lead to S-nitrosylation of mitochondrial proteins to cause energy depletion⁶⁴. Additionally, IL-1β from inflammasome-activated macrophages activates NF-kB and p38 MAPK, which further increase RNS^65,66. Importantly, H₂O₂ may fuel the Fenton reaction to induce lipid peroxidation and ferroptosis⁵⁶. And besides the canonical TNF-α/TNFR1 pathway, DAMPs such as high mobility group box-1 (HMGB1) are involved in I/R-induced inflammation and cause necroptosis in cardiomyocytes⁸. Beyond the inflammatory signaling, although the removal of dead cardiomyocytes by macrophage phagocytosis is necessary for heart repair, it is not clear if the phagocytotic process has a direct impact on the survival of the residual cardiomyocytes.

4.3. Protective Roles of Macrophages after MI:

Maintaining a homeostatic energy metabolism in cardiomyocytes is essential for tissue repair after MI. Resident cardiac macrophages can form a network to support mitochondrial homeostasis by taking up and clearing “unfit” mitochondria from cardiomyocytes^31,67. This process of mitochondrial transfer is a non-immune function that helps to preserve the metabolic stability and cardiac function. Depletion of the C-X3-C Motif Chemokine Receptor 1 (Cx3cr1)-based TIMD4⁺ resident macrophages leads to impaired cardiac function and promotes adverse ventricular remodeling in the peri-infarct zone⁶⁷. To support cardiomyocyte viability, resident macrophages produce IGF-1, NRG-1 and OSM, which act as survival signaling for cardiomyocytes^44,68. NRG1 also enhances mitochondrial function to maintain energetic homeostasis in cardiomyocytes¹. Other factors, including IL-10, TGF-β and Prostaglandin E2, can be produced by reparative macrophages to suppress destructive inflammatory signal, hence indirectly promote cardiomyocyte survival¹⁴.

On the cellular level, it is not clear if the physical proximity between macrophages and cardiomyocytes confer protection to the latter. Macrophages connect with nodal cells (specialized cardiomyocytes) through gap junctions to facilitate electrical conduction²⁹. Similarly, cardiac fibroblasts with high level of ferritins establish gap junctions with adjacent cardiomyocytes to share iron burden and prevent ferroptosis⁷. In mouse models of I/R, ferritin iron accumulation was also observed in macrophages⁸, it would be informative to investigate if macrophages can protect adjacent cardiomyocytes through a similar mechanism. Should one subset of cardiac macrophages possess superior iron-storing and -metabolizing capacity compared to other subsets, it can be targeted to enhance the iron-handling ability of the myocardium to protect cardiomyocytes from ferroptosis.

4.4. Impact of sex differences on immune cells and cardiomyocyte death after MI:

Sex difference is a major factor in the outcomes of patients with cardiovascular diseases (CVDs). Historically known to impact more male than female, CVD is now a top killer of female in the U.S., with higher death rates compared to male patients⁶⁹. Epidemiological factors including health habits, diabetes, hypertension, and atypical presentation (in women) often complicate the interpretation of clinical data and translation of basic science findings³. Pre-menopause women have lower risk of MI than age-matched men due to lower risk factors such as hypertension⁷⁰. The risk of MI in women increases significantly after menopause, emphasizing the roles of estrogen signaling. Clinical trials supported this hypothesis, showing significant cardiovascular benefits in younger patients who initiated hormone replacement therapy early in the perimenopausal period⁷¹. Estrogen, mainly 17β-estradiol (E2), directly benefits cardiomyocytes by triggering survival signaling such as the PI3K/Akt and MAPK, enhancing mitochondrial function, reducing ROS and inhibiting NF-kB. The result is decreased apoptosis, necrosis and ferroptosis in cardiomyocytes^70,72.

Despite a lower risk of heart attack, young women (≤55 years) with acute MI have higher risk of mortality caused by cardiac rupture than age-matching male patients^70,73. Two emerging hypotheses include: 1) Young women may experience higher mortality due to differences in diagnostic standard which can be improved by public education and clinical studies. This is supported by mouse studies in which young females have higher survival rates and decreased LV dilation during acute phase after MI⁷⁰; 2) Young women may have a higher inflammatory state after acute MI. While estrogen is protective, women have other risk factors such as higher level of high-sensitivity C-reactive protein (hsCRP), a marker of systemic inflammation associated with increased cardiovascular risk and recurrent cardiovascular event⁷⁴. The extra X chromosome, although partially inactivated, can still affect inflammation. In mice, X chromosome promoted cardiac injury due to elevated level of inflammatory genes (escapees of X chromosome inactivation) such as IL-1 receptor-associated kinase 1 (IRAK1) and Toll-like Receptor 7 (TLR7)^70,75,76.

Sex differences in MI involve distinct immune responses. Immune cell population in female hearts present a broad spectrum of activation status compared to a more polarized pattern in male heart after MI⁷⁰. Estrogen has been linked to decreased leukocyte accumulation after I/R injury as reviewed by DeLeon-Pennel et al.⁷⁰. After acute MI, more neutrophils have been shown to infiltrate the infarct zone in male and become more active and release higher level of TNF-α and IL-6^77,78. Although having less hyper-active cell populations during acute inflammatory phase can be beneficial, a robust sterile inflammation facilitates the formation of mature scarring and prevents myocardial rupture. The stoichiometric impact of different immune cells makes it difficult to isolate the effect of macrophages and define their roles. But certain immune cell subsets such as the blood CD64⁺CD169⁺TIMD4⁺ monocytes expand after ST-elevation MI in both men and women, analogous to mice model²⁶. Such monocyte/macrophage population can be targeted for therapeutic purpose to avoid treatment discrepancies caused by sex differences.

5. Precision Therapy and Future Efforts

Based on current knowledge of macrophage function in MI, therapeutic strategies focus on amplifying the beneficial signaling derived from macrophages and/or inhibiting excessive inflammation. NRG1 and IGF-1 are now in clinical trials for treating heart failure. Anti-TNF-α/IL-1β therapies such as canakinumab are also promising for improving cardiomyocyte viability⁷⁹. ROS scavengers such as N-acetyl cysteine and mitoTEMPO can protect against oxidative damage⁸⁰. However, the redundancy of macrophage-derived cytokines suggests that using single factor or antagonist may not be sufficient⁴³. Therefore, boosting polarization of macrophages to a reparative phenotype via IL-4/IL-13 delivery⁷⁹ or IL-19 treatment⁵⁹ could be an alternative. The heterogeneity of macrophages hinders the efficiency of non-specific immunosuppressive strategies in clinical trials. Effective methods would harness the healing power of reparative macrophage subsets, such as the Trem2^high population, and prevent maladaptive fibrosis simultaneously. Alternatively, CCR2⁺ monocytes can be programmed to be less inflammatory and more reparative at appropriate pathological stage. For example, the nuclear receptor Nr4a1 is vital for balancing the inflammatory and reparative phases after MI. Targeting the Nr4a1 pathway in Ly-6C^high monocytes could help limit the influx of inflammatory monocytes and promote a more reparative macrophage phenotype¹⁵.

Other plausible approaches include reforming the progenitor source of macrophages. Targeting splenic reserves with liver X receptor-α (LXR-α) agonist can expand the beneficial marginal zone macrophages, that alleviate inflammation and improve cardiac repair²⁶. The CH also inspires the design of interventional approaches. Both MI and CH are age-related, and CH is a risk factor for coronary heart disease²². Maintaining the HSC quiescence with active metabolites of vitamin A (e.g. 4-oxo-retinoic acid) effectively safeguards HSCs from activation by MI, therefore dampens inflammatory myelopoiesis and preserves cardiac function⁸¹. The CH driven by mutations in driver genes, such as the Tet Methylcytosine Dioxygenase 2 (TET2) (loss-of-function), promotes cardiac infiltration of macrophages and increases myocardial inflammation⁸². Tet2-deficient macrophages produce more IL-1 and IL-6 compared with macrophages without Tet2 mutations²². Interestingly, a genetic mutation in IL6R, that results in reduced IL-6 signaling, is associated with reduced risk of cardiovascular disease in individuals with TET2-induced CH⁸³. These data suggest IL-6-IL6R signaling axis as a potential target for treating heart attack in patients with CH.

Despite intense efforts and progress, regenerating the myocardium after MI remains the “holy grail” of modern cardiovascular research. Due to the limitation of the current discussion, we cannot fully appreciate the complexity of the spatiotemporal roles of sterile immune system after MI. Therapeutic strategies are constantly inspired by information acquired from clinical studies and mammalian heart models of MI. However, the versatile roles of immune cells in cardiac repair are sometimes contradictory. The survival or death signal from macrophages can be magnified or suppressed through interaction with other immune cell types. The functions and mechanisms of the same subset of macrophages may vary at different developmental stages, likely due to the different intrinsic strategies of cardiac repair in neonatal (cardiomyocyte proliferation) and adult (fibrotic remodeling) heart. Thinking reversely, the impact of MI on the origins of macrophages, including the HSC and spleen, also needs further investigation⁸⁴. Meanwhile, as more evidence emerging to show that cardiomyocytes are the primary initiator of a pathological innate immune response in the infarct border zone³³, it is necessary to interrogate the impact of cardiomyocytes on macrophages and other immune cells when undergoing different type of RCDs.

Moreover, the difficulty to translate knowledge acquired from mouse models to clinical therapies is also prominent. Findings in rodent models will likely need to be validated in large mammals. Last but not least, the thought that a specific RCD is more regenerative for cardiomyocytes is intriguing. Hence preventing a certain type of RCD in cardiomyocytes when the stress is still present may not be beneficial for the heart, as they may switch to an alternative RCD and produce a wrong group of paracrine factors⁵⁹.

Highlights.

1. Myocardial infarction (MI) causes severe inflammation and cardiomyocyte death.

2. Besides necrosis and apoptosis, cardiomyocytes in the infarct zone undergo ferroptosis, necroptosis, pyroptosis etc.

3. Macrophages have diverse ontogeny and pathological functions, exhibiting versatile, yet contradictory roles on the survival and death of cardiomyocytes.

4. The insight of the roles of macrophages in cardiomyocyte survival and death can guide the design of therapies for treating MI.

Nonstandard Abbreviations and Acronyms

CCL2

C-C chemokine ligand 2

CCR2

C-C Motif Chemokine Receptor 2

CD163

scavenger receptor cysteine-rich type 1

CH

clonal hematopoiesis

CVD

cardiovascular disease

Cx3cr1

C-X3-C motif chemokine receptor 1

DAMP

damage-associated molecular pattern

E2

17β-estradiol

ECM

extracellular matrix

FOLR2

folate receptor beta

Fuca1

alpha-L-fucosidase 1

GSDMD

gasdermin D

HMGB1

high mobility group box 1

HSC

hematopoietic stem cell

hsCRP

high-sensitivity C-reactive protein

Ifit

interferon induced protein with tetratricopeptide repeats

IFN-γ

interferon gamma

Igf-1

insulin-like growth factor 1

Il1b

interleukin 1 beta

IL-6

interleukin-6

I/R

ischemia reperfusion

IRAK1

IL-1 receptor-associated kinase 1

Irf7

interferon regulatory factor 7

Isg20

interferon stimulated exonuclease gene 20

LAD-O

left anterior descending coronary artery occlusion

Ly-6C

lymphocyte antigen 6 complex C

LYVE1

lymphatic vessel endothelial hyaluronan receptor 1

LXR-α

liver X receptor-α

MHC-II

major histocompatibility complex class II

MI

myocardial infarction

MLKL

mixed lineage kinase domain like pseudokinase

NLRP3

NOD-like receptor 3

NO

nitric oxide

Nrg1

neuregulin 1

Nrp1

neuropilin 1

ONOO^-

peroxynitrite

Osm

oncostatin M

PL-PUFA

polyunsaturated acyl tail

Pltp

phospholipid transfer protein

RCD

regulated cell death

RIPK1

receptor interacting serine/threonine kinase 1

RNS

reactive nitrogen species

ROS

reactive oxygen species

Serping1

serpin family G member 1

SOCS3

suppressor of cytokine signaling 3

TET2

tet methylcytosine dioxygenase 2

TGF-β

transforming growth factor-β

TIMD4

T cell immunoglobulin and mucin domain-containing 4

TLR7

toll-like Receptor 7

TNF

tumor necrosis factor