The Role of Neuregulin and Stem Cells as Therapy Post-Myocardial Infarction

The Use of Stem Cells for Therapy Post-MI

The discovery of endogenous stem cells within cardiac tissue has incited a fury of research around potential regenerative therapies for cardiac pathologies, such as congestive heart failure (CHF) and MI [9]. As with any potential stem cell therapy, a variety of approaches have undergone testing, and several have proven successful. Totipotent embryonic stem cells often present the best option, as these cells have the greatest potential to become any cell toward which they are molded. Embryonic stem cells are one of the few stem cell types that have been shown to electrically link up with endogenous cardiac cells after engraftment [22].

In 2014, Chong and Murry demonstrated, among nonhuman primate hearts, that human embryonic stem cell-derived CMs can not only be used to engraft and remuscularize large areas of infarcted myocardium but also can be produced at a scale that is viable for clinical use [22]. Soong et al. put forth that embryonic and embryonic-like stem cells can be used to derive a therapeutic substitute myocardium, for these cells can be cultured reliably and robustly to produce CMs [23,24]. However, the usage of embryonic stem cells is riddled with ethical questions. In addition, preclinical animal trials have indicated an increased risk of teratoma development [23,24]. This risk poses a great obstacle, particularly with regard to human trials and the progression of these trials into advanced clinical phases [9,25].

Bone marrow MSCs have been shown to promote myocardial regeneration in animal models post-MI, but it is unclear if these stem cells are directly contributing to CM regeneration or aiding in regeneration by supporting angiogenesis. Notably, bone marrow MSCs can differentiate into fibroblasts, contributing to the negative remodeling effects post-MI [9]. In light of this body of research, we believe that embryonic stem cells may offer the best opportunity for clinical and therapeutic success in the repair of damaged cardiac tissue post-MI, given their ability to generate cells with cardiomyogenic properties at a clinically significant scale. Notably, the techniques for harvesting these cells and applying them in humans still require further elucidation and consideration of short- and long-term clinical challenges.

There has been great success with engraftment of cardiac stem cells into animal models with subacute MI using cardiac stem cells generated from human induced pluripotent stem cells (hi-PSCs). Ma et al. found that the low differentiation of iPSC-derived CMs limited their therapeutic applications, which prompted them to experiment with the addition of electrical stimulation during the differentiation of the stem cells [26]. They found that preconditioning of electrical stimulation significantly increased the therapeutic efficacy of cardiac stem cell therapy in a post-MI heart.

Electrical stimulation increased the number of cardiac progenitor cell genes (Nkx2–5 and GATA4) and structural genes (ɑ-myosin heavy chain) expressed. It additionally led to an increased number of beating embryonic bodies [26]. Electrical stimulation also helped induce the engrafted hi-PSC CMs to form striations and to better mimic their surrounding environment [26].

To test the efficacy of electrical stimulation in an animal model, hi-PSCs were engrafted into a mouse model mimicking a subacute MI. Cells that were pretreated with electrical stimulation had higher rates of engraftment and function compared to the control hi-PSCs that did not receive electrical stimulation [26]. Similarly, other studies have found that co-culturing the iPSCs with a particular miRNA profile (miR125b, miR199a, miR221, and miR222) can help the iPSC-derived CMs achieve a proper mature structure with the organization and bulk of adult CMs [27,28].

Furthermore, Masumoto et al. found that using VEGF instead of Dk11 (a canonical Wnt antagonist) induced greater differentiation of iPSCs into endothelial cells and mural cells, both of which are important supporting cells of CMs, both in culture and in vivo [29]. They found that hi-PSCs treated with VEGF showed greater cTnT (titin) expression and that they formed a structure that more closely mirrored that found in endogenous human hearts [29].

To test the efficacy of VEGF in inducing proper cardiac structure in hi-PSCs, they engrafted the cells into a rat model that had undergone occlusion of the left anterior descending artery (LAD) to simulate a subacute MI. By tracking the engrafted cells with human nuclear antigen, they found that the hi-PSCs treated with VEGF showed greater CM engraftment, even many weeks after the surgery [29]. They also found that there was a significant increase in capillary density, as shown by greater staining for von Willebrand factor. These findings suggest that culturing the hi-PSCs with VEGF has a positive angiogenic effect on the infarcted area [29].

In an alternative study, a protocol utilizing different concentrations of activin A and bone morphogenetic protein 4 (BMP4) was employed to polarize hi-PSCs into high-purity mesodermal subtypes reflecting cardiogenic and hemogenic mesoderm, allowing for distinct progenitor populations. With greater understanding of mechanisms involved in deriving these cell types, next steps can be taken toward generating CMs for therapeutic use [22].

In recent years, important advances have been made to understand the application of stem cells in engineering cardiac tissue. Zimmermann et al. have developed a novel method that relies on collagen and extracellular matrix-based materials [30]. The resulting engineered heart tissue possesses the functional and morphological properties of differentiated CMs, making it a potential tool for replacing damaged myocardium following MI or heart failure [30,31].

The authors state that for this to be a clinically viable tool, the stem cell resource should be autologous, available in adequate quantities, and of course, demonstrate characteristics of cardiac tissue. They proposed the use of embryonic and certain adult stem cells due to their capacity for proliferation. However, limitations include the ability to produce the stem cell-derived CMs at large enough quantities.

In an effort to resolve this issue of efficiency, later work conducted by Tiburcy et al. sought to advance CM maturation in engineered human myocardium from embryonic and iPSC-derived CMs and fibroblasts toward an adult phenotype [31]. With this protocol, the investigators were able to generate heart muscle that is differentiated and force-generating, thus having important implications for heart repair. The advanced maturation was noted, as the heart muscle could support mechanical and electrical stimulation. In its totality, this body of research provides proof-of-concept for the engineering of human myocardium from embryonic and iPSC-derived CMs that can be utilized in disease modeling as well as in heart repair [30,31].

There has also been further testing of the efficacy of iPSCs in larger animal models, such as monkeys. Ishigami et al. used iPSCs that had been induced in culture to form sheets through temperature modulation. They found that implanting these sheets restored left ventricle (LV) function in monkeys with induced LV MI [32]. In addition, they noted less fibrosis and increased spontaneous beating along with calcium movement within the cells [32].

While many of these studies were limited by their modeling of the MI or their use of animal models, there are ongoing trials using stem cell treatment in humans (Table 1) [33 –36]. Herreros et al. enrolled 12 patients with old MI injuries, who were undergoing coronary artery bypass surgery to receive intramyocardial injections of autologous skeletal myoblast cells in conjunction with the surgery; they found a significant increase in LV ejection fraction (LVEF) and regional contractility, demonstrating the utility and feasibility of stem cell injections intraoperatively for patients with damaged cardiac tissue [33].

In a similar trial, patients who were a few days status-post reperfusion of acute MIs were subject to the intracoronary injection of either autologous bone marrow-derived stem cells or circulating blood-derived progenitor cells [34]. Assmus et al. found that both patient populations that received progenitor cell injections had significant increases in LVEF and improved contractility in the infarcted area compared to a nonrandomized control group [34].

There are other ongoing randomized studies testing both autologous and allogeneic cardiac-derived stem cell injections for improvement of cardiac function post-MI as well as proving the safety of the method (ALLSTAR and CADESCEUS trials) [35,36]. While these studies are still in the preliminary phase, and many more clinical trials are needed, they have demonstrated the potential for the therapeutic effects of cardiac stem cells. Furthermore, these results can lead researchers to explore the utility and efficacy of the combined use of stem cells and NRG-1 to further aid in the regeneration of cardiac tissue post-MI.

In addition, there are many ongoing trials using stem cells in humans that are still in their infancy and have not yet published results (Table 2). There are trials investigating the safety and efficacy of the use of MSCs derived from Wharton's Jelly in umbilical cord tissue for improving cardiac function post-MI [37 –41]. Furthermore, there is a clinical trial in the recruiting phase to use a biologic drug (MiSaver^®) as a treatment for MI [42]. There is also an ongoing trial to investigate the efficacy and safety of autologist PB-CD34+ stem cells in patients with acute MIs [42]. While none of these trials has published results yet, the continued interest and work in this field of stem cells as a treatment for MIs show that much progress has been made toward finding an effective treatment.

Emerging Use of NRG-1 for Therapy Post-MI

There is currently a growing body of literature documenting the crucial role of NRG-1 and its signaling receptors in cardiac development as well as in the maintenance of cardiac function throughout adulthood in both physiological and pathological states [11,43,44]. Research has demonstrated that NRG-1 not only has direct effects on CMs but also acts within the vasculature, interstitium, cardiac fibroblasts, hematopoietic cells, and immune cells.

Within these cells, NRG-1 is implicated in CM survival, the differentiation of embryonic stem cells into cardiac cells, cell migration, angiogenesis, cytoskeletal assembly, excitation coupling, formation of the neuromuscular junction, and structural components of the cardiac interstitial matrix [12,44,45]. As NRG-1 is involved in many aspects of cardiac development and function, its use as a therapy in patients following MI, and in subsequent heart failure, is of significant interest.

The clinical burden of disease associated with MI and subsequent heart failure drives interest in finding a means to stimulate CM regeneration following ischemic injury. D'Uva et al. conducted a study in neonatal, juvenile, and adult mice to demonstrate the role of ErbB2 signaling and its effects in cardiac tissue [46]. The murine neonatal heart is capable of regeneration following injury due to CM proliferation resulting from upregulated expression of ErbB2. However, expression is reduced shortly after birth.

Given this observation, a constitutively active ErbB2 was induced and was found to result in cardiomegaly characterized by remarkable hypertrophy, dedifferentiation, and proliferation of cells [46]. When the same constitutively active receptor was transiently induced in the murine population post-MI, it similarly triggered dedifferentiation, neovascularization, and proliferation of the CMs. Overall, these results suggest that upregulation of the NRG-1/ErbB2 signaling can lead to anatomical and functional cardiac regeneration following injury. However, overexpression of the ERB2 receptor subunit may promote uncontrolled cancer growth [47]. Through ERB2 inhibition, trastuzumab has been used to treat some of these cancers, especially those arising in the breast [47].

NRG-1 has also been employed as a tool in pharmacological ischemic postconditioning (IP) [48]. IP is a strategy used following MI to reduce infarct size that involves cycles of ischemia and reperfusion at the site of infarction. In a study of myocardial reperfusion injury in a rat model, the cardioprotective effects of NRG-1 were evaluated in comparison to traditional IP. The results revealed that NRG-1 had a similar effect to IP and was able to reduce the size of infarct post-MI and reduce apoptosis [48].

NRG-1 is not only capable of assisting in CM regeneration but also may have effects on sympathetic and vagus nerve remodeling post-MI. One study randomized 45 rats to (1) surgical exposure of the LAD without ligation, (2) LAD ligation to simulate MI, or (3) LAD ligation followed by intraperitoneal administration of NRG-1 daily over a period of 7 days [44]. Four weeks post-MI, rats receiving NRG-1 showed significant improvements in cardiac function, as assessed by increased LVEF, increased LV fractional shortening, and reduced nervous remodeling.

These findings are significant, given that sympathetic hyperinnervation is likely to occur following ischemic injury and can potentially lead to ventricular arrhythmia [44]. NRG-1 can thus be used to improve cardiac function and to simultaneously reduce sympathetic nerve tension post-MI, consequently reducing the risk of arrhythmia [44].

NRG-1 has a number of protective effects, which suggests that its disruption can also increase susceptibility to prolonged damage following injury to myocytes, such as in the case of MI and heart failure [43]. Exogenous administration of NRG-1 has been shown to improve cardiac function in patients with heart failure. In a 2014 study, NRG-1β was administered to a population of rats following LAD ligation to simulate MI [45]. Following administration, subjects were noted to have improved cardiac function and reduced interstitial fibrosis, even when treatment was delayed to 2 months post-MI [45].

Another study sought to build on these findings by examining administration of exogenous NRG-1 in a population of rats induced with type 1 diabetes mellitus (T1DM). This population is of particular interest in application to humans since individuals with T1DM post-MI are more susceptible to heart failure. In these cases of heart failure, NRG-1/ErbB signaling is notably impaired [43,45].

Results of the study show that administration of NRG-1 is capable of mitigating progression of heart failure, improving cardiac remodeling, suppressing myocardial fibrosis and apoptosis, and reducing oxidant-producing enzymes [43]. These findings suggest that NRG-1 can not only be used to mitigate the damaging effects of cardiovascular ischemia and heart failure in a general population but also can be applied to a population with comorbidities, such as T1DM. Further study in populations with other comorbidities, such as T2DM and obesity, is warranted.

As clinical trials are ongoing, one concern for translating treatment into a clinical setting could be determining dosing that is practical for patients. One study looked to address this concern by assessing different dosing regimens of recombinant NRG-1, referred to as GGF2, and its effect on LV function in rats with surgically induced MI [12]. Results demonstrated that GGF2 treatments beginning 1 to 2 weeks post-MI can improve LVEF and other aspects of LV function. These improvements were greater with increased dose, and were shown to have greater improvements when treatment was delivered infrequently [12]. Moreover, improvements were sustained over 40 weeks even with just once-weekly or every 2-week dosing [12]. Given the promising effects of this infrequent dosing, GGF2 is likely to be a practical candidate for recombinant NRG-1 heart failure therapy in the future.

Research into the use of NRG-1 for cardiovascular regeneration and repair has predominantly focused on exogenous administration. However, two studies have experimented with the endogenous upregulation of NRG-1/ErbB2 signaling by exercise training. One study found that when rats were subject to 4 weeks of exercising following LAD ligation to simulate MI, NRG-1 was upregulated [14]. Exercise therapy promoted cardiac repair and regeneration, while improving angiogenesis and reducing apoptosis [14]. Several rats were additionally subjected to ErbB2 inhibition. Inhibition of ErbB2 signaling attenuated exercise therapy-induced cardiac repair and regeneration, suggesting that NRG-1/ErbB2 signaling plays a crucial role in cardiovascular regeneration [14].

A similar study subjected rats to 4 weeks of resistance training, consisting of moderate-intensity and high-intensity intermittent aerobic exercise [49]. This study also found that exercise upregulates expression of NRG-1 and promotes heart function, reduces cardiac fibrosis, increases muscle weight, and increases cross-sectional area of muscle fibers [49]. Survival rates post-MI were also improved in rats that underwent resistance training [14]. These findings similarly suggest that NRG-1 is upregulated following exercise training and plays an important role in cardiovascular function post-MI.

While much of this research on the use of NRG-1 as a therapy for patients post-MI and with heart failure has been conducted in animal models, two clinical trials are ongoing in human subjects [11]. One phase II, randomized, double-blind trial has currently enrolled 44 patients with CHF characterized by LVEF ≤40%. IV recombinant human NRG-1 (rhNRG-1) administered 8 h per day for 10 days showed an improvement in LVEF of 32%, compared to an improvement of 15% in patients receiving placebo. Another single-center, prospective, nonrandomized, open-label study showed that when rhNRG-1 was administered to patients with CHF for 6 h per day over 11 days, there were rapid improvements in cardiac function, marked by improved cardiac output, stroke volume, and mean arterial pressure [11].

The results of these studies are promising and have strong implications for the use of NRG-1 both independently and in combination with other agents, such as stem cells, in the treatment of cardiovascular disease.

Combinatorial Use of Stem Cells and NRG-1 for Therapy Post-MI

Given the advancement in the applications of both NRG-1 and stem cells as therapeutic agents post-MI, researchers are beginning to explore their possible synergistic effects. In 2013, Díaz-Herraez et al. investigated the combinatorial use of NRG-1 and adipose-derived stem cells (ADSCs) using a mouse MI model involving the permanent occlusion of the LAD artery [50]. In this study, NRG-1-releasing particle scaffolds were adhered to ADSCs and injected around the border of the infarct zone. By encapsulating the NRG-1 into a scaffold, the researchers hoped to create a delivery system by which NRG could be released in a sustained manner, while providing protection from degradation [50].

Following successful encapsulation and implantation, the scaffolds were found to be well tolerated and integrated in the host tissue, where they remained detectable at 2 weeks postimplantation [50]. This study therefore serves as a proof of concept for the potential use of a unique delivery system for multiple growth factors and cytokines that could be beneficial post-MI. As noted by the authors, further experimentation is necessary to determine the effectiveness of this approach as a therapy [50]. Furthermore, the use of stem cells in the protein scaffold was largely for the beneficial paracrine effects they provide on the endogenous cardiac cells, and there was no intent to induce the ADSCs to differentiate into a cardiac lineage [50].

Additional research has focused on the potential of stem cells to become functional CMs. However, despite this enormous interest in the regenerative potential of stem cells in the heart, research thus far has shown a largely protective rather than regenerative effect [18]. This is due, in part, to barriers to transplantation of progenitors that effectively differentiate into mature cardiovascular cells with the full functionality of an intrinsic CM [51].

A 2014 study hypothesized that the use of NRG-1β could be used to differentiate iPSCs in vitro into more functional and mature CMs than previously achieved, thereby reducing the risk of immunogenicity and tumorigenesis of in vivo differentiation, while maximizing the potential for regeneration [18]. Using a mouse model, this experiment demonstrated that treatment of iPSCs in vitro with NRG-1β works synergistically with dimethyl sulfoxide (DMSO) to allow for differentiation into effective ventricular-like cardiac cells. Importantly, the researchers concluded that treatment with NRG-1β/DMSO created CMs with electrophysiology similar to that of neonatal CMs [18]. Upon transplantation into the mouse model of acute MI, the iPSCs were able to synchronize electromechanically, reducing the risk of cardiac arrhythmias [18].

Although the mechanisms by which NRG-1β assists in this more complete and successful differentiation of iPSCs are not yet fully understood, the transplantation of this population of iPSCs in the mouse model following cardiac ischemia showed decreased deleterious cardiac remodeling and increased revascularization of tissue [18]. Taken together, this allowed for an overall superior cardiac functionality of the experimental group compared to the control mice that had received iPSCs grown in media without NRG-1β/DMSO [18]. Despite these results, the authors acknowledged that the in vivo CM engraftments in this experiment were “only temporary or low degree,” and proposed that further research focus on strategies to increase the likelihood of long-term stem cell survival post-transplantation [18].

These obstacles may be overcome with the use of an alternate cell population, such as MSCs. In addition to the use of MSCs instead of iPSCs, Liang et al. further sought to determine if the use of genetic modification of the MSCs themselves could be used to simultaneously increase the likelihood of stem cell survival and have a therapeutic effect in the post-MI setting [19]. Also using a mouse model, the researchers transplanted NRG-1 alone, MSCs alone, or MSC-ErbB (MSCs into which they had transduced ErbB4) into the mice post-MI. The latter experimental group allowed for the overexpression of ErbB4 by the MSCs [19].

The results of this study were positive in multiple important ways. First, their hypothesis concerning the increased viability of stem cells post-transplantation proved true, as the MSC-ErbB group showed improved MSC survival [19]. This was especially impressive considering it came at no cost to functionality; in fact, the transplantation of the MSC-ErbB cells resulted in smaller infarct size, less apoptosis, increased mitosis of CMs, and preserved ventricular function compared to the other groups. The authors further discovered a previously unknown autocrine loop in the MSC-ErbB cohort, wherein the overexpression of ErbB4 resulted in NRG-1 upregulation. This allowed for the replenishment of NRG-1 in ischemic areas of the heart, where it is typically deficient [19].

Additional experimentation by Liang et al. reinforced the potential beneficial effect of ErbB4 overexpression on MSCs post-MI, particularly by activation of PI3K/AKT and MAPK/ERK pathways [20].

These results reinforce the potential of the combinatorial use of NRG-1 and stem cells to bring about regeneration following MI.

Conclusion

The growing epidemic of cardiovascular disease and rates of MI have motivated researchers to investigate alternative therapies for use post-MI. Following an MI, cardiac tissue undergoes apoptosis and inflammation, potentially resulting in the formation of scar tissue and the dysregulation of electrical circuits in the heart leading to arrhythmias [1]. Current pharmacological treatments are preventive measures, but they fail to restore the full function of the cardiac tissue. With the escalating number of patients with cardiovascular disease, there is a strong need for alternative regenerative therapies.

Therapeutic use of stem cells has been tested for a long period of time, but comes with limitations as well. Success has been seen with the engraftment of cardiac stem cells in subacute MI animal models using hi-PSCs. The results of these studies have pushed researchers to test these stem cells in human subjects; ongoing and future trials will provide further information on the therapeutic effects of cardiac stem cells.

NRG-1 signaling has proved to be an essential component for cardiac development and regeneration, making it a potentially useful alternative therapy in the context of cardiovascular disease. The protective effects of NRG-1 are remarkable, as increased expression reduces susceptibility to ischemic damage [43]. When constitutively active signaling was induced in murine populations post-MI, upregulation of signaling led to anatomical and functional cardiac regeneration [46]. These effects can be seen in the dedifferentiation of cells, CM proliferation, reduced nervous system remodeling, and increases in LVEF [11,43,46].

The results seen with upregulation of NRG-1 in study populations post-MI have been overwhelmingly positive. As future trials focusing on the use of NRG-1 move to study the effects in human populations, efforts should be made to further study the impact in patients with comorbidities associated with cardiovascular disease, such as T2DM and obesity.

Despite the promising application of stem cells and NRG-1 as separate therapies post-MI, there seems to be greater excitement and potential with the combinatorial approach. Studies have found that NRG-1 and iPSCs can act synergistically to decrease cardiac remodeling and improve LV function in mice models. However, it remains unclear which type of stem cells would be optimal to use with NRG-1. Studies have shown that NRG-1 and MSCs act optimally together, allowing for increased stem cell viability, reduced infarct size, reduced apoptosis, increased CM proliferation, and preserved LV function. However, It is important to note that other research has shown that, in isolation, MSCs may actually promote negative remodeling and fibrosis post-MI [9,19].

Currently, research examining the possible synergistic effect of NRG-1 with hi-PSCs cultured in VEGF is lacking. When used alone, hi-PSCs treated with VEGF showed excellent long-term CM engraftment and increased angiogenic activity postinfarction [29]. Notably, Iglesias-Garcia et al. found that when iPSCs were treated with NRG-1β and DMSO in vitro, there was reduced deleterious cardiac remodeling, improved revascularization, and reduced risk of tumorigenesis and immunogenicity. However, the CM engraftment in this set of experiments was only temporary [18].

In reflecting on the work of Masumoto et al. and Iglesias-Garcia et al., it is possible that the combination of NRG-1 and hi-PSCs treated with VEGF could overcome the challenges of temporary engraftment, while also showing excellent improvements in overall cardiac function and angiogenesis.

In consideration of the available evidence, we believe such a combination therapy involving the use of stem cells and NRG-1 may be a turning point in regenerative medicine. The use of these agents together requires further research to elucidate a combination of stem cells and NRG-1 that maximizes both efficacy and safety. Combining the beneficial effects of both stem cells and NRG-1 will likely lead to therapeutic advancements for patients with cardiovascular disease, such as those who are post-MI or those experiencing associated heart failure.