Numerous factors can influence tissue regeneration, including oxygen stress, immune response, sympathetic and parasympathetic signaling, ECM, metabolism, and various micro RNAs (miRNAs) (69–72). In previous studies, it has been shown that regeneration of salamander limbs is blocked when macrophages are deleted, and amputated limbs do not regenerate if the severed end is covered by fibrotic tissue. When the loss of macrophages was reversed by restoring macrophages, salamanders regained the ability to regenerate body parts (73). In addition to limb regeneration, Godwin et al. also reported an indispensable role for macrophages in salamander heart regeneration (74), consistent with the results of Aurora's studies (59). These studies have shown that macrophages activate fibroblasts and regulate lysyl oxidase activity soon after heart ischemic or cryogenic injury (59, 74). In later studies using gene editing, such as Tribbles homolog 1, or drug delivery, such as clodronate liposomes, it was shown that macrophage deficiency is lethal to mammals and increases the risk of cardiac rupture (75–78).

Monocytes derived from myeloid progenitors are also an important component of the immune system owing to their potential to differentiate into dendritic cells and macrophages. They can be recruited from bone marrow and extramedullary hematopoietic sites, migrating to specific destinations in response to chemokine binding to CCR2 (79). In general, monocytes in the mouse can be divided into two subsets, classical [expressing high lymphocyte antigen 6 complex (Ly6C), positive for CCR2 and low in chemokine C-X3-C motif receptor 1 (CX3CR1)] and non-classical (expressing low Ly6C, negative for CCR2 and high in CX3CR1) (36). The corresponding subsets in humans are a cluster of differentiation (CD) 14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes (79, 80). After an ischemic injury, abundant circulating Ly6C^high (CD14⁺⁺CD16⁻) monocytes are rapidly recruited to the infarcted area, where they contribute to the initiation of neutrophil infiltration and differentiate into macrophages that remove cell debris and degrade the extracellular matrix (79, 81). In the later stage of the response, Ly6C^low (CD14⁺CD16⁺) monocytes, which can be derived from Ly6C^high monocytes (81), tend to accumulate in the area and express high levels of growth factors like vascular endothelial growth factor (VEGF) (36), contributing to tissue healing (81). Nuclear receptor subfamily four group A member 1 (Nr4a1) is crucially important for the development of Ly6C^low monocytes and regulation of monocyte behavior and cytokine expression by Ly6C^high monocytes (81, 82). In a clinical study on the association between different monocyte subsets and prognosis of patients with acute myocardial infarction (AMI), it was found that CD14⁺⁺CD16⁻ monocytes were negatively related to the myocardial repair and recovery of left ventricular function (83).

In general, macrophages are crucial for repair after injury. During tissue repair, macrophages can aggravate an ischemic injury by disturbing the microenvironment and metabolism and causing cell death. On the other hand, cytokines like vascular endothelial growth factor-α (VEGF-α), insulin-like growth factor-1 (IGF-1), produced by macrophages promote the proliferation of various cells (84).

In terms of heart regeneration and repair, macrophage functions can be divided into three primary categories (Figure 1). First, macrophages can stimulate the proliferation of endothelial cells, smooth muscle cells, and fibroblasts by releasing growth factors (85, 86) and promoting angiogenesis for tissue restoration. In the study of Fantin et al., cardiac tissue macrophages were shown to participate in the formation of capillary networks in the myocardium, which might be mediated by the neuropilin1 signaling pathway. Following this, macrophages can work as chaperones in tip cell fusion targeted by vascular endothelial growth factor (VEGF) (85). Moreover, it has been reported that macrophages can differentiate into endothelial cells or endothelial precursors (87, 88).

Second, ECM composition is regulated by macrophages. In an axolotl cryogenic cardiac model, researchers found that fibrosis was disrupted when macrophages were depleted, showing that macrophages are crucial for fibrosis and maturation of the ECM (74). On the one hand, by secreting TGF-β and fibroblast growth factors, macrophages indirectly activate myofibroblasts to produce collagen and work together with them to release matrix metalloproteases (MMPs) that modify ECM composition (84, 89). On the other hand, many studies have discovered that macrophages can directly promote fibrosis. In 1999, Weitkamp et al. first found macrophages, isolated from human samples, could secrete Type VIII collagen in vitro (90). Later, other studies also identified that macrophages could synthesize proteoglycans and related proteins (91), Type VI collagen (92, 93), which were important components of ECM. In a research published in 2020, by analyzing the transcriptome from zebrafish hearts following injury (1 day, 5 days, and 14 days) and comparing P1 and P7 neonatal mouse hearts 7 days after myocardial infarction, collagen and ECM-associated genes were found in macrophages, indicating their autonomous and direct role in fibrosis (94). In mice, macrophages produced about 6.8% of total collagen (94). Since macrophages could exhibit a fibrosis-associated phenotype, some researchers proposed that macrophages could go through a fibroblast-like transition after ischemic injury (93, 95, 96).

Third, macrophages can stimulate cardiomyocytes to dedifferentiate and enter the cell cycle (25). In neonatal humans and mice, it was revealed that resident macrophages play an important role in augmenting cardiac proliferation under low oxygen conditions (97). In salamanders, however, when macrophages were depleted in a heart cryo-injury model, whole heart regeneration was blocked when the activation of fibroblasts and remodeling of the ECM was disturbed. In contrast, cardiomyocyte proliferation was unaffected (74). Given these disparate results, the ability of macrophages to directly or indirectly promote cardiomyocyte proliferation in mice and humans requires further study.

When the heart is injured, different types of macrophages usually respond in a variety of ways over different time scales (98). In the early response to ischemic damage signals in mouse, numerous Ly6C^high monocytes are recruited from blood and lead to further M1-type macrophage activation, which provides the injured area with a “spring-cleaning” (36, 99, 100). In addition, M1-type macrophages can significantly upregulate MMP1, 3, 7, 10, 14, and 25 to modulate the ECM (41, 81, 101). Meanwhile, Ly6C^high monocytes and other immune cells, mainly neutrophils, secrete a series of chemokines and cytokines like IL-1β, IL-6, tumor necrosis factor (TNF)-α, and chemokine (C-C motif) ligand (CCL)-3, and CCL-4 to amplify inflammation and activate cardiomyocytes and endothelial cells (102). At later times during tissue repair, changes in the microenvironment stimulate macrophages to polarize toward the M2 type and to increase the concentration of VEGF, and myeloid-derived growth factor (MYDGF) (75, 103), which increases cell proliferation and production of new vessels (23, 59, 78). In some organs, M2 macrophages seem to be more essential for repair than the M1 type (75), and in the heart, a reduced M1/M2 ratio is associated with increased numbers of vessels and more stable ECM. However, contrary to earlier results, a recent study reported simultaneous pro-inflammatory and anti-inflammatory effects in response to cardiac cryoinjury, but not a transition from M1 to M2 macrophages (104). Also, crosstalk between M1 and M2 macrophages should not be neglected as M1 polarization may be essential for the activation of M2 (46, 105). Since lipopolysaccharide (LPS) and IL-4 can elicit different types of macrophages in vitro, Munoz-Rojas et al. co-stimulated macrophages with LPS+IFN-γ and IL-4, and by single-cell sequencing, they found that this kind of macrophage contained a new transcriptome. Interestingly, some cytokines like IL-6, IL12b, arginase-1 (Arg1), and chitinase-3-like protein 1 (Chi3l1) began to be exclusively expressed in macrophages (106).

As we discussed earlier, macrophages can be classified as resident or monocyte-derived. These two kinds of macrophages have distinct functions as well (47, 48, 107). Normally, both resident and monocyte-derived macrophages exist in neonatal and adult hearts, but their subtypes and quantities differ. Neonatal hearts, which can renew themselves, will selectively amplify the resident population with little increase in the numbers of monocyte-derived cells (Figure 1). In contrast, adult hearts contain increased numbers of monocyte-derived macrophages, although the percentage of resident cells is initially similar to neonates (30, 60, 63). In a cardiomyocyte ablation experiment, it was found that resident macrophages could produce less inflammation and promote cell proliferation and the formation of new coronary vessels, while monocyte-derived macrophages only had inflammatory effects (60). Also, it was shown that macrophage amplification following injury in neonatal mice occurred rapidly, over no more than 5 days, while inflammation and repair tended to be slower in adult hearts (60, 97). However, since monocyte-derived macrophages can replace the loss of resident cells, the differences between responses to injuries in neonatal and adult hearts require further study to elucidate mechanisms.

In ischemic heart disease, different responses are observed in infarct, peri-infarct, and remote regions (49). In the infarct zone, the number of resident macrophages is greatly reduced and numerous monocytes are recruited for the clearance of cell debris. In the peri-infarct zone, the rapid proliferation of resident macrophages contributes to myocardial repair, the loss of which can lead to negative remodeling. Finally, in the remote zone, little proliferation or change in the numbers of resident macrophages is observed. After an injury, CCR2– and CCR2+ resident macrophages contribute to the recruitment of monocytes (47). CCR2+ macrophages promote monocyte and neutrophil recruitment through damage-associated molecular patterns (DAMPS)-toll-like receptor (TLR)-myeloid differentiation factor 88 (MyD88) signaling, but in contrast, CCR2– macrophages may reduce the aggregation of monocytes (47, 108).

Macrophages play various roles in non-ischemic heart diseases, such as pressure overload hypertrophy and chronic heart failure. In a hypertension model where macrophages were depleted by clodronate liposomes, loss of heart function and fibrosis were clearly attenuated (109). When mechanical strain is exerted on the myocardium, the mitogen-activated protein kinase (MAPK) pathway can be activated, promoting re-entry to the cell cycle and increasing the proliferation of macrophages (63). Pressure-overload hypertrophy is often accompanied by the activation of sympathetic nerves and angiotensin-family proteins. It has been shown that the CXCL1-CXCR2 axis participates in the regulation of monocyte recruitment during hypertrophy (110). In the early stages of hypertrophy, resident macrophages proliferate under the influence of the transcription factor krüppel-like factor 4 (KLF4), leading to an increase in the formation of new vessels (49, 111). Monocytes then take over the role of the resident macrophages to function in cardiac protection (49).

Since macrophages play a crucial role in heart regeneration, various factors affect the process. Cells like neutrophils (112), Foxp3+CD4+ regulatory T cells (113), cytokines like IL-4 (75), IL-35 (114), IL-10 (33, 105, 115, 116), other proteins like Dectin-2 (98), matrix metalloproteinase-28 (117), and serum-glucocorticoid regulated kinase 1 (118) can all affect the activation of macrophages or the M1/M2 ratio, and thus likely influence heart regeneration. Nuclear receptor subfamily 4 group A member 1 (NR4A1) inhibits the expression of IL-6 and TNF to promote phenotype transitions of macrophages. In recent years, great progress has been made in revealing metabolic transformations in macrophages. It has been reported that changes in energy sources, such as glucose or lipids, can affect the utilization of substrates by macrophages, as can macrophage polarization (46, 119, 120).

Given the crucial role of macrophages in cell proliferation and heart regeneration, many studies seek new targets for stimulating macrophages to more efficiently exert positive effects. Some studies have reported that anti-TNF therapy had a certain effect on cardiac function in tumor patients with heart failure (121), thus demonstrating an effect of specific anti-immune therapy. Clinically, many drugs, such as trimetazidine or sodium-glucose cotransporter-2 (SGLT2) inhibitors and anti-inflammatory therapies, such as IL-1β inhibition are proven to have an effect on improving cardiac function (46, 122–124). However, whether they may target macrophages is uncertain. In recent years, many studies have tried to investigate the potential therapy methods, such as simple compounds, nanoparticles, exosomes from macrophages (engineered and unprocessed), and mesenchymal stem cells (MSCs), targeting macrophages during cardiac injury (both ischemic and non-ischemic injuries) in animal models (77, 125–130). Compared to simple compounds, nanoparticles and engineered exosomes provide for more precise targeting, thus decreasing the side effects and enhancing the effectiveness (25). In a recent study, Rangasami et al. used nanoparticles derived from hyaluronic acid (HA) to deliver anti-cancer drugs, strengthening the effectiveness while avoiding side effects (130). This offers a new therapeutic tool as the contents of the particles can be changed to make full use of the HA shell. However, considering the fact that many markers and characteristics of macrophages are known from experiments in vitro, the current classification of macrophages may not allow for precisely targeting specific subsets that exist in vivo. In addition, the tissue environment is not the same for mice and humans. Therefore, more basic research and clinical studies are needed to explore therapeutic strategies.