Introduction
Cardiovascular diseases (CVDs) remain the leading cause of death world-wide, accounting for 31% of all fatalities in 2016 (WHO, June 2016). An important contributing factor is our incomplete understanding of the processes by which tissue is remodeled after a myocardial infarction (MI). One hallmark of the disease is the recruitment of a diverse range of immune cells which are recruited into the infarcted heart and modulate both innate and adaptive immune responses [
111]. During the initial phase after MI, for example, inflammation plays a causal role in remodeling the left ventricle (LV) and is accompanied by a rearrangement of myocytes, extracellular components and vessels [
67].
Infiltrating cells exhibit specific patterns of spatiotemporal distribution and activity [
212] while carrying out an active, sequential crosstalk with each other and other cardiac cells. This creates a highly complex regulatory landscape [
132,
142] that plays an important role in proper post-MI cardiac healing [
149]. Inflammatory processes may also cause hypertrophy, fibrosis and other types of cardiac damage which can subsequently lead to heart failure [
102]. Optimum recovery, thus, requires a timely and selective modulation of inflammation [
48,
49], but the heterogeneity and functional diversity of immune cells pose challenges in attempts to target inflammation as a therapeutic strategy. Successful approaches will require a more comprehensive understanding of the spatiotemporal coordination of immune responses in post-MI tissue.
Here, we highlight the temporal dynamics of immune cells during post-MI LV wound healing and consider the therapeutic potential of engineering such cells. We focus particularly on recent findings regarding the roles of noncoding RNAs (ncRNAs) in regulating immune cell functions. An additional focus is the growing list of molecules known to participate in the recruitment, activation and polarization of immune cells after MI, opening new avenues for pharmaceutical manipulation that may lead to improved forms of immunotherapy for MI patients.
Summary: non-coding RNAs and immune regulation
Mounting evidence regarding the many diverse ways noncoding RNAs (ncRNAs) serve as master regulators of gene expression in diverse situations involving immunity and wound healing have brought these molecules to attention as potential targets for therapies. Studies have shown that ncRNAs are expressed in a highly lineage-specific manner and regulate the differentiation and function of innate and adaptive immune cells—both of which are crucial in attempts to develop therapies that target pathological processes with high specificity in the environment of post-MI cardiac tissues. A global disruption of immune cell types would almost invariably have negative consequences on patient health, particularly in the context of a dynamic tissue in which the roles of inflammatory and adaptive immune cells change over time.
A recent study from Halade et al. showed that miRNAs play a role in regulating gene expression related to leukocyte kinetics following MI. This suggests that modulating the MI-coordinated miRs could provide hints towards the regulation of post-MI inflammatory responses [
62]. Wang et al. have reported a cardio-protective role for miR-146a. Its transfection inhibited the activation of NF-kB and diminished the infiltration of neutrophils into the heart following myocardial I/R, leading to reduced infarct size and improved cardiac function following MI [
201]. Lowering the expression of another miRNA, miR-223, was associated with an increase in neutrophil infiltration and myocardial dysfunction in a sepsis patient via activation of STAT3/IL6 and Sema3, indicating that the presence of the miR-223 prevents this influx and lowers inflammation [
202]. miR-21 and -150 also help prevent adverse MI remodeling through their effects on leukocyte numbers and subsequent vascular inflammation [
11,
18,
113]. Deficiency of miR-21 in macrophages promotes apoptosis-related signaling pathways, including the MKK3/p38 and JNK pathways, leading to apoptosis and vascular inflammation during atherogenesis [
18]. miR-150 negatively regulates expression of the chemokine receptor 4 (CXCR4) which in turn induces monocyte migration, thereby decreasing infiltration of inflammatory monocytes and improving cardiac function, as shown in miR-150 overexpressing mice [
113]. A significantly reduced expression of miR-144 is associated with improper cardiac remodeling, while restoring endogenous levels of myocardial expression of miR-144 through intravenous injections improves post-MI cardiac function. Additional mechanistic studies have demonstrated that miR-144 inhibits inflammatory and auto-phagocytic signaling pathways, indicating that miR-144 might have its beneficial effects by lowering the infiltration of macrophages and improving autophagy [
106].
MiR-155 expression is significantly and primarily upregulated in macrophages, in the post-MI myocardium; interestingly, levels differ between M1 and M2 macrophages. Its depletion promotes M2 polarization and improves cardiac function following viral myocarditis [
219]. This suggests that miRNA-155 might serve as a prognostic marker for cardiac death in post-MI patients [
123]. miR-155 is also found in exosomes released by macrophages and this has effects on fibroblasts, which in turn trigger a dysregulation of fibrosis [
197]. miR-155 is mainly involved in B- and T-cell receptor signaling, neurotrophin signaling, MAPK signaling, and the cell cycle. Especially in regard to the cell-cycle signaling pathway, Sos1 expression is increased in the absence of miR-155, and is associated with fibroblast proliferation post-MI [
197]. Also, angiotensin II-induced expression of the angiotensin II type 1 receptor (AT1R) and extracellular signal-related kinase 1/2 (ERK1/2) are downregulated by miR-155 [
123]. Overall, the inhibition of miR-155 activity seems to have therapeutic potential in seeking to minimize post-MI cardiac injury [
197].
Circulating miR-133 and miR-33 directly promotes macrophage polarization and has further effects on lipid metabolism, as seen in the myocardial steatosis that develops in type 2 diabetes patients [
34,
143]. miR-33 expression levels have been used as diagnostic marker for diabetic cardiomyopathy [
34]. miR-33 mediates anti-inflammatory macrophage polarization by targeting the energy sensor AMP-activated protein kinase (AMPK) pathway [
143]. Two further miRNAs, miR-150 and miR-181a, play roles in regulating both DC differentiation and vascular inflammation. Necrotic cardiomyocyte-stimulated DC maturation requires the JAK1-STAT1/c-Fos pathway, concomitant with decreased miR-150 and increased miR-181a levels. Modification of these miRNAs, either through the overexpression of miR-150 or through the inhibition of miR-181a, respectively, downregulates DC maturation and leads to a reduction in the apoptosis of cardiomyocytes, indicating a potential therapeutic approach to preserve cardiomyocytes after a cardiac injury such as MI [
225].
Various lncRNAs have also been implicated in immune regulation (Table
2). Vausort et al. showed a strong connection between the inflammatory response and lncRNAs including hypoxia inducible factor 1A antisense RNA 2 (aHIF), cyclin-dependent kinase inhibitor 2B antisense RNA 1 (ANRIL), MI-associated transcript (MIAT) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Their expression was closely associated with blood cell count as well as the abundance of neutrophils and lymphocytes. Cardiovascular risk factors such as aging or diabetes boost their levels even higher. So far, a direct connection to post-MI cardiac dysfunction has only been established for ANRIL [
190]. Levels of the myocardial infarction-associated transcript-1(Mirt1) and Mirt2 are elevated in MI and peak at 24 h post-MI, strongly suggesting another case of ncRNAs linked to inflammatory regulation [
215]. LPS-induced Mirt2 regulates inflammatory cytokine production through the polarization of macrophages towards a M2 phenotype via suppression of NF-κB and MAPK pathways [
39]. Mirt2 overexpression protects against endotoxemia-induced mortality and organ dysfunction [
39]. Furthermore, both Mirt1 and Mirt2 target cardiac remodeling genes such as mmp-9, Icam1 and tgfb1 during post-MI wound healing [
215]. The upregulation of both lncRNAs have been negatively correlated with post-MI cardiac remodeling, accompanied by the smaller size of infarcts and better ejection fractions, indicating that elevations in Mirt1 and Mirt2 expression balance the inflammatory response and preserve cardiac function post-MI [
215].
Table 2
Non-coding RNAs as a biomarker and therapeutic approaches in myocardial infarction
Inhibition of inflammation | miRNA | miR-144 | ↓Pro-inflammatory response | N/A | ↓ |
miR-146a | ↓Infiltration of neutrophils, infarct size | Cardiomyocytes | ↑ |
miR-150, -181a | ↓DCs maturation, cardiomyocyte apoptosis | Dendritic cells | ↑ |
miR-223 | ↑Neutrophil infiltration, inflammation | Cardiac muscles | ↓ |
Let-7i-5p | ↓Inflammatory cytokine production, fibrosis | Fibroblasts | ↑ |
lncRNA | ANRIL | Increase blood cell count, associated with cardiac dysfunction | PBMCs | ↑ |
LINC00305 | Accelerated monocyte-mediated inflammation | PBMCs | ↑ |
LncRNA-1055, -A930015D03Rik | ↑Th1 mediated immune response, ↑cardiac inflammation | N/A | N/A |
Resolution of inflammation | miRNA | miR-133 | Pro-inflammatory macrophage polarization, lipid metabolism | Macrophages | ↑ |
miR-155 | Macrophage polarization, ↑fibroblast proliferation ↑Treg proliferation | Macrophages | ↑ |
lncRNA | Mirt1, -2 | Macrophage polarization, ↑cardiac function | Fibroblasts | ↑ |
Modulation adoptive immunity | lncRNA | LncRNA-E330013P06 | ↑Foam cell production, ↑atherosclerosis | Macrophage | ↑ |
The inflammatory response post-MI can result in systemic atherosclerosis with elevated numbers of macrophage-derived foam cells accompanied by enhanced lipid metabolism [
85]. LncRNA E330013P06 is expressed at high levels in foam cells, a phenomenon associated with exaggerated cardiac inflammation. Sustained E330013P06 levels are correlated with elevated levels of pro-inflammatory genes and pro-atherogenic genes, which contribute to foam cell formation. Inhibiting the expression of E330013P06 in macrophages reduces the production of both foam cells and the expression of inflammatory genes in diabetes patients [
152]. There are still crucial questions regarding the E330013P06 underlying molecular mechanism that controls the cardiac inflammatory response, which merits further studies. A further pro-inflammatory lncRNA, LINC00305, is also highly expressed in monocytes derived from patients with atherosclerosis and is associated with an exaggerated inflammatory response [
217]. LINC00305 promotes an interaction between the membrane protein lipocalin-interacting membrane receptor (LIMR) and the inflammatory gene aryl hydrocarbon receptor repressor (AHRR) via activation of the NF-kappaB pathway in human monocytes, further contributing to the development of atherosclerosis [
217]. These findings suggest that LINC00305 could be a novel target for an anti-inflammatory therapy. LncRNAs can also regulate autoimmunity, as the expression of lncRNA-A930015D03Rik and -1055 is strongly correlated with IL12Rβ1, one of the essential molecular markers in Th1 response pathway. Knocking down lncRNA-A930015D03Rik and -1055 to modulate the Th1-mediated immune response and cardiac inflammation is an interesting line of future therapeutic strategies [
56].
A function in the context of immunity has not been described for most ncRNAs, although dramatic changes in ncRNA expression have been clearly shown during the activation of immune cells. This further strengthens the argument that ncRNAs can act as immune regulators and should therefore not be considered mere transcriptional ‘noise’. Further investigations into ncRNAs and their potential immune functions will undoubtedly yield insights into the mechanisms that balance the inflammatory response, which could ultimately lead to improved treatments for cardiovascular diseases and other pathologies.
Non-coding RNA based therapeutics
An increasing number and range of functions are being found for ncRNAs in processes related to the dynamic development, function and activation of cells, all of which are relevant to pathologies. This has given rise to the concept of manipulating disease-related signaling pathways by targeting cell-specific ncRNAs in developing new approaches to therapies. The main aim of such ncRNA-based therapies has generally been to alter abnormal levels of expression of ncRNAs by restoring them to the basal level. In the investigation of ncRNA functions, antagonists and ncRNA mimics are being effectively used; most therapeutic efforts are based on these strategies as well. [
154]. Antisense technologies which sequester or degrade mature ncRNAs are currently the most efficient approaches in silencing ncRNA activity.
miRNAs seem to be particularly targetable through the delivery of reverse complimentary anti-miRNA oligonucleotides, which function by either sequestering the target molecule or by triggering their degradation by cellular RNA interference mechanisms. Antagonists usually need to be modified to enhance their stability and improve binding efficiency, through chemical modifications such as the addition of 2-
O-methyl groups, or methylene linkers that ‘lock’ the oligonucleotides in a more robust conformation (LNA) [
179]. Additionally, cholesterol-conjugated antagomirs might be useful tools, as demonstrated by Krutzfeldt et al. who achieved a cardiac tissue-specific miRNA knockdown by injecting the compounds into mice [
98]. The LNA-based approach is currently being tested in an anti-miRNA therapy in stage IIa of clinical trials, using an inhibitor designed to target miR-122 in cases of chronic Hepatitis C infection. This study represents a good example how to translate a miRNA-focused therapy in the clinic, and it suggests that targeting immune cell-derived miRNAs in the heart is a practicable strategy.
Another concept that has emerged in altering miRNA expression involves “sponges”, which do not actively trigger their degradation but rather serve as baits that prevent their binding to target mRNAs. Such sponges may be constructed to target multiple miRNAs and have longer lifespans than miRNA inhibitors. Wang’s group has designed a ‘Multiple Target AMO Technology (MT-AMO)’, in the form of a single-stranded, methyl-modified oligonucleotide sequence capable of binding multiple miRNAs within a single family of seeds or even multiple families [
203]. This could be useful in treating human pathologies, including cardiovascular diseases, in which several miRNAs are deregulated.
The novel CRISPR/cas9 system has also been used to knock-down expression of ncRNAs [
20,
26]. Chang et al. demonstrated that the CRISPR approach not only reduces the off-target seen with miR inhibitors or mimics, but also has a knock-down effect on miRNAs that is sustained much longer in both in vitro and in vivo models [
20]. Synthetic ncRNAs and individual ncRNAs transduced using viruses are the most common method of restoring levels of downregulated ncRNAs [
194], but the results are not always reproducible or very potent, making efforts to re-constitute miRNA levels lag behind those aimed at depleting them.
Our review shows that levels of expression of an increasing number of ncRNAs are now known to change following MI [
190]. Recent work has deepened our understanding of the way the immune regulation of non-coding RNAs influence post-MI cardiac functions, particularly where the inflammatory response is concerned. The findings open ncRNAs to new approaches for clinical translation in efforts to achieve optimal post-MI wound healing.
Conclusion and future direction
Dynamic immune responses regulate key events during post-MI cardiac repair. Distinct types of immune cells act in precisely timed ways and take on diverse roles in preserving cardiac function after MI. Improper or imbalanced immune responses have adverse effects on LV remodeling and enhance the progression to heart failure. Understanding these cell fates and functions and the diverse factors related to immune action will be required to help develop an improved microenvironment that encourages repair. A number of approaches to regulate the immune response following MI damage are under development. The first step in moving these concepts into clinical practice is to understand how ncRNAs regulate the functions of the immune cell repertoire in achieving a balanced inflammatory response in the context of the post-MI heart. Given that key molecules are expressed in different cell types, often with contradictory functions at different time points, applications will require a very profound understanding of the cell-specific functions of ncRNAs and the way they change over time.
Data are particularly needed on the way the origin and spatiotemporal distribution of these immune cells function in the broader context of immunity, which has not been documented here. To date, most experimental models have been limited because they are based on models where age, gender and genetic background are highly standardized. This is never sufficient in characterizing the progression of diseases that affect patients who are mostly older and have co-morbidities [
141]. What will be needed is a “higher resolution” view of the kinetics of immune cells over time in different strains of animals, particularly during dynamic inflammatory states in areas of the heart and body beyond those directly affected by an infarction. While much has been learned about the dynamics of immune cells that closely associated with cardiac dysfunction in the ischemic myocardium, much less is known about inflammatory changes in remote post-MI myocardium. There is evidence that remote myocardium dysfunction indirectly or directly contributes to functional and morphological changes in the infarct region [
15]. To thoroughly understand the systemic immune response following MI, further studies will be needed that focus not only on ischemic lesions, but also on non-ischemic lesions; they will need to be carried out in a wider range of experimental models with some connection to cardiovascular physiology. Another issue will be to characterize the crosstalk between immune cells, other cardiac cells and those in other tissues. There is every sign that addressing these gaps in our knowledge will identify fruitful new avenues toward diagnosing, treating and preventing MI, as a means of improving the lives of the growing number of patients suffering from this dreaded disease.