Cardiovascular Research

Cardiovascular Research 2000 48(1):89-100; doi:10.1016/S0008-6363(00)00158-9
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb)

Nikolaos G. Frangogiannis^*, Lloyd H. Michael and Mark L. Entman

Section of Cardiovascular Sciences, Baylor College of Medicine and the De Bakey Heart Center, The Methodist Hospital, Houston TX, USA

^* Corresponding author. Tel.: +1-713-798-4188; fax: +1-713-796-0015 ngf{at}bcm.tmc.edu

Received 2 March 2000; accepted 6 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Objective: The purpose of this study is to examine the cellular content of healing myocardial infarcts and study the phenotypic characteristics of fibroblasts during scar formation utilizing a canine model of coronary occlusion and reperfusion. Methods: Ischemia/Reperfusion experiments were performed in dogs undergoing 1 h of coronary occlusion followed by reperfusion intervals ranging from 5 h to 28 days. Fibrotic and control areas were studied using immunohistochemistry. Results: The healing ischemic and reperfused myocardium demonstrated significant proliferative activity peaking after 3 to 7 days of reperfusion, predominantly in myofibroblasts. The numbers of proliferating cells decreased during the maturation phase of the scar (PCNA index: 13.7±2.25% at 5 days vs. 4.8±1.1% at 28 days; P<0.05, n = 5). During the proliferative phase of healing (3–7 days) -smooth muscle actin (-SMAc) expression was markedly increased in the fibrotic areas. -SMAc predominantly localized in myofibroblasts which were vimentin positive, smooth muscle myosin, calponin and desmin negative. We examined expression of smooth muscle myosin heavy chain isoforms in myofibroblasts infiltrating the healing areas and found a marked induction of the embryonal isoform of myosin heavy chain (SMemb) in -SMAc positive spindle shaped cells in the border of the scar. Myofibroblasts did not express SM2, a marker for mature smooth muscle cells. In contrast myocardial arterioles were positive for SM2, but did not express SMemb. Conclusions: Healing myocardial infarcts undergo rapid changes in their content of myofibroblasts. During the proliferative phase fibroblasts undergo phenotypic changes leading to expression of contractile proteins such as -SMAc, and production of SMemb, a marker for dedifferentiated smooth muscle cells. Expression of embryonic isoforms indicates dedifferentiation and allows the myofibroblast pool to serve as a versatile cell population, assuming different phenotypes depending on the physiological needs.

KEYWORDS Fibrosis; Histo(patho)logy; Infarction; Reperfusion; Smooth muscle

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Wound healing is a dynamic biological process, requiring the collaborative efforts of many different cell types [1–6]. Scar formation involves a series of rapid increases in specific cell populations that prepare the wound for repair, deposit new matrices and finally, lead to scar maturation [1–6]. Macrophages, lymphocytes and mast cells infiltrate the healing tissue [7–10] and provide a source of cytokines and growth factors necessary to stimulate fibrosis [11] and angiogenesis [12,13]. Fibroblasts produce the extracellular matrix constituents needed to support cell ingrowth and newly formed blood vessels carry oxygen and nutrients necessary to sustain cell metabolism [1–6,8].

Myocardial ischemia and reperfusion is associated with an intense inflammatory reaction leading to healing of the injured myocardium [14–16]. Mononuclear cells and mast cells accumulate in the ischemic heart (9,10) and may serve as an important source of cytokines and growth factors [14]. Studies utilizing a variety of experimental models [10,17–19] have demonstrated the presence of -SMAc positive myofibroblasts [20–23] in the healing myocardium. These cells were found to be capable of active proliferation [10]. In this study we sought to investigate the phenotypic characteristics of fibroblasts in the infarcted canine myocardium. We identified myofibroblasts as -smooth muscle actin and vimentin positive cells and found that they do not express smooth muscle myosin, desmin and calponin. We present the first demonstration of myofibroblasts expressing the embryonal isoform of smooth muscle myosin (SMemb/non muscle myosin heavy chain B) located in the border zone of reperfused myocardial infarcts. Expression of SMemb, a marker for dedifferentiated smooth muscle cells emphasizes the phenotypic plasticity of the myofibroblast population, which may be capable of fulfilling a variety of distinct functions, including angiogenesis and matrix formation.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
2.1 Ischemia/reperfusion protocols
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85–23, revised 1996). Healthy mongrel dogs (15–25 kg) of either sex were surgically instrumented as previously described [10,24,25]. Anesthesia was induced intravenously with 10 mg/kg methohexital sodium (Brevital, Eli Lilly and Co, Indianapolis IN) and maintained with the inhalational anesthetic isoflurane (Anaquest, Madison, WI). A midline thoracotomy provided access to the heart and mediastinum. Subsequently, a hydraulically activated occluding device and a Doppler flow probe were secured around the circumflex coronary artery just proximal or just distal to the first branch. Choice of location depended on the proximity and anatomical arrangement. Indwelling catheters placed in the right atrium, left atrium and femoral artery allowed blood sampling and pressure monitoring as needed. After surgery, the animals were allowed to recover for 72 h before occlusion. Coronary artery occlusion was achieved by inflating the coronary cuff occluder until mean flow in the coronary vessel was zero as determined by the Doppler flow probe. At the end of 1 h, the cuff was deflated and the myocardium was reperfused. Reperfusion intervals ranged from 5 h to 28 days. Circumflex blood flow, arterial blood pressure, heart rate and ECG (standard limb II) were recorded continuously. Analgesia was accomplished with intravenously administered pentazocine (Talwin; Winthrop Pharmaceuticals, New York, NY) 0.1–0.2 mg/kg. After the reperfusion periods, hearts were stopped by the rapid intravenous infusion of 30 meq of KCl and removed from the chest for sectioning from apex to base into four transverse rings 1 cm in thickness. The posterior papillary muscle and the posterior free wall were identified. Tissue samples were isolated from infarcted or normally perfused myocardium based on visual inspection. Myocardial segments were fixed in 10% buffered formalin, or B*5 fixative [26] or snap frozen for histological analysis. Duplicate adjacent samples were also processed for blood flow determinations using radiolabeled microspheres as previously described [10,23]. Thirty-five dogs with myocardial infarction were included in the study (five experiments for each reperfusion interval).

The presence of a myocardial infarct was based on light-microscopic examination of hematoxylin-eosin-stained tissue sections by findings of contraction bands, "wavy fibers", interstitial edema and neutrophil infiltration, all in segments displaying markedly reduced blood flow (<25% control) during the ischemic period. For experiments lasting 24 h or more after the start of the ischemic insult, the presence of histological elements characteristic of myocyte necrosis and fibrosis was added to the required criteria. Samples described as ischemic were all from areas where blood flow was less than 25%, as determined by the radiolabeled microspheres method. Samples of control tissues were taken from the anterior septum and had normal blood flow during coronary occlusion.

2.2 Immunohistochemistry and histology
For histological study of cardiac tissue, sections taken from endocardium to epicardium and sections taken parallel to the wall of the heart were fixed in 10% phosphate buffered formalin, or B*5 fixatives and embedded in paraffin. Immunohistochemical studies were performed as previously described [25,27], using samples fixed in B*5 fixative, which ensures optimal antigenic survival. Sequential 3–5 µm sections were cut by microtomy. Immunostaining was performed using the ELITE rabbit or mouse kit (Vector Laboratories, Burlingame CA). Briefly, sections were pretreated with a solution of 3% hydrogen peroxide to inhibit endogenous peroxidase activity and incubated with 2% goat serum to block nonspecific protein binding. Subsequently, they were incubated with the primary antibody for 2 h at room temperature. After rinsing with PBS, the slides were incubated for 30 min with the secondary antibody. The slides were rinsed with PBS and incubated for 30 min in ABC reagent. Peroxidase activity was detected using diaminobenzidine (DAB) with nickel. Slides were counterstained with eosin. Frozen sections were immunostained using the same protocol after brief (5 min) fixation in absolute ethanol.

The following primary monoclonal antibodies were used for immunohistochemistry: anti-PCNA antibody (Dako) to identify nuclei of proliferating cells, anti CD31 antibody (Dako) to label endothelial cells, anti -SMAc antibody (Sigma), anti-vimentin antibody (Sigma), anti-calponin antibody (Sigma), anti smooth muscle myosin antibody [28] (kindly donated by Dr. Seidel, Baylor College of Medicine, Houston TX), anti-desmin antibody (Sigma), anti-SM1, anti-SM2 and anti-SMemb antibodies [29–32] (Yamasa Corp). Staining for SM1 was successful in human cardiac biopsies, but not in canine tissues suggesting no crossreactivity of the antibody with canine species. SM2 and SMemb staining was successful only in frozen sections. All other antibodies gave good immunohistochemical results when used in B*5 fixed paraffin embedded canine tissues. Sections incubated with nonimmune serum were used as negative controls. In addition, sections from normal canine bowel were used as positive controls for staining with the antibody to PCNA.

2.3 Quantitative analysis of immunohistochemical experiments
At least two ischemic and two control segments were studied from each experiment. Stained sections were photographed with a Leaf MicroLumina digital camera mounted on a Zeiss microscope. Multiple digital images were taken and stored for each sample. Staining was analyzed by Zeiss image-analysis software. The PCNA index was quantitated by expressing the number of PCNA positive nuclei as a percentage of the total number of hematoxylin stained nuclei in a field.

2.4 Statistical analysis.
The statistical significance of the findings was assessed by ANOVA. This was followed by a Student's t-test, corrected for multiple comparisons (Bonferrroni). In histological studies each experiment and time point was analyzed as a function of time of reperfusion after 1 h of occlusion. The findings described all occurred in at least three consecutive experiments

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 PCNA expression following myocardial ischemia and reperfusion
Immunohistochemistry with an antibody to the Proliferating Cell Nuclear Antigen (PCNA) (Fig. 1) showed significantly higher numbers of proliferating cells in the ischemic and reperfused myocardium after 1 h of ischemia and 72 h of reperfusion when compared to shorter reperfusion intervals (PCNA index 11.3±1.53% at 72 h vs. 0.56±0.33% at 5 h; P<0.05, n = 5). The PCNA index peaked at 5 to 7 days of reperfusion and decreased following 21 to 28 days (13.7±2.25% at 5 days vs. 4.8±1.1% at 28 days; P<0.05, n = 5). PCNA positive nuclei were very rare in control canine myocardium. In a previous study [10], we identified the majority of PCNA positive cells as -SMAc positive myofibroblasts.

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Cell proliferation following myocardial ischemia/reperfusion. Time course of the PCNA index in ischemic areas following experimental myocardial infarction. Note that proliferative activity increases after 72 h of reperfusion (PCNA index 11.3±1.53 at 72 h vs. 0.56±0.33 at 5 h; P<0.05, n = 5), peaks following 5 to 7 days and decreases after 21 to 28 days (PCNA index 13.7±2.25 at 5 days vs. 4.8±1.1 at 28 days; P<0.05, n = 5). Symbols: * P<0.05 when compared to PCNA index at 5 h, **P<0.05 when compared to PCNA index at 5 days. A (insert): Photomicrograph demonstrating PCNA positive nucleus (arrowhead) in a fibrotic region after 28 days of reperfusion. Counterstaining with hematoxylin shows the nuclei of non-proliferating cells.

 
3.2 Microvessel density in the healing scar
CD31 positive microvessels (Fig. 2A) were counted in healing myocardial scars following various reperfusion intervals. Microvessel density tended to increase with scar maturation peaking at 21 days of reperfusion (3808.5±443.5 mv/sq mm in fibrotic areas vs. 2066±107.9 mv/sq mm in nonfibrotic areas; P<0.05, n = 5) (Fig. 2).

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Microvessels in the healing reperfused canine myocardium. Microvessel density during maturation of the healing myocardial scar. Microvessel numbers peak after 21 days of reperfusion (3808.5±443.5 mv/sq mm in fibrotic areas vs. 2066±107.9 mv/sq mm in nonfibrotic areas; *P<0.05, n = 5). A (insert): Photomicrograph showing CD31 positive microvessels in a fibrotic area.

 
3.3 Alpha-smooth muscle actin expression in the healing myocardium.
Immunohistochemical experiments with an antibody to -SMAc identified myofibroblasts in the ischemic and reperfused myocardium (Fig. 3A–D). In normal canine myocardium -smooth muscle actin was predominantly expressed in vascular medial smooth muscle cells. There was a trend towards increased -SMAc staining after 72 h of reperfusion. The percent area stained for -SMAc in fibrotic areas peaked after 5 to 14 days of reperfusion (fibrotic at 5 days 12.94+2.25% vs. non-fibrotic 2.42+0.66%; P<0.05, n = 5). Alpha smooth muscle actin staining decreased following 21 to 28 days of reperfusion reflecting the decreasing numbers of myofibroblasts in the maturing scar (fibrotic at 28 days 4.07+0.76% vs. fibrotic at 5 days 12.94+2.25%; P<0.05, n = 5) (Fig. 3E).

View larger version (202K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Alpha-smooth muscle actin expression in the healing canine myocardium. A–D. Immunostaining for -SMAc following 7 days (A), 14 days (B), 21 days (C) and 28 days (D) of reperfusion. Significant numbers of -SMAc positive myofibroblasts are found in the healing scar after 7 to 14 days of reperfusion. E: -SMAc peaked after 5 to 14 days of reperfusion (fibrotic at 5 days 12.94+2.25% vs. non-fibrotic 2.42+0.66%; P<0.05, n = 5) and decreased with scar maturation (fibrotic at 28 days 4.07+0.76% vs. fibrotic at 5 days 12.94+2.25%; P<0.05, n = 5). Symbols: *P<0.05 when compared to control areas from the same experiment, **P<0.05 when compared to fibrotic area at 5 days of reperfusion, when maximal -SMAc expression is noted.

 
3.4 Phenotypic characteristics of myofibroblasts in healing myocardial scars:
Utilizing immunohistochemical techniques we demonstrated that medial smooth muscle cells in canine coronary arteries and arterioles express SM2, -SMAc, desmin, calponin. Furthermore, smooth muscle cells in the coronary arteries, but not in the myocardial arterioles express SMemb. Following myocardial ischemia and reperfusion myofibroblast-like cells expressing -SMAc appear in the healing myocardium. These cells produce -SMAc and vimentin, but not smooth muscle myosin, calponin (Fig. 4) and desmin (Fig. 5). They have proliferative capacity (10) and their numbers increase during the proliferative phase of healing. During the maturation phase -SMAc staining decreases reflecting decreased myofibroblast numbers, which likely become apoptotic.

View larger version (148K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Phenotypic characterization of myofibroblasts in the healing myocardium (1 h ischemia/72 h reperfusion) Immunohistochemistry for vimentin (A), -SMAc (B), smooth muscle myosin (C) and calponin (D). Note that infiltrating myofibroblasts are nonvascular -SMAc positive cells (arrows), which express vimentin (A), but not smooth muscle myosin (C), or calponin (D). In contrast, smooth muscle cells in the media of the arteriole (arrowhead) produce both smooth muscle myosin and calponin.

 

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Myofibroblasts in the ischemic and reperfused canine heart do not express desmin (representative experiment of 1 h ischemia, 7 days reperfusion). A: Immunohistochemistry for desmin. B: Staining for -SMAc: Note that the -SMAc positive myofibroblasts located in the fibrotic area do not stain for desmin. In contrast, some of the arteriolar vascular smooth muscle cells are desmin positive. Cardiomyocytes also demonstrate intense staining for desmin.

 
3.5 Myofibroblasts express the embryonal isoform of smooth muscle myosin heavy chain (smemb)
Staining of control areas of canine heart demonstrated that myocardial arterioles express -SMAc and SM2, a marker for mature smooth muscle cells, but not SMemb. In healing myocardial infarcts a large number of spindle shaped myofibroblast-like cells express SMemb (Fig. 6). These cells are also positive for -SMAc but do not express SM2. SMemb positive myofibroblasts are predominantly located in the border zone of healing myocardial infarcts (Fig. 6).

View larger version (129K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Myofibroblasts in the healing scar express SMemb and not SM2. Immunohistochemistry in frozen sections using antibodies to SMemb (A, B, D), -SMAc (C), and SM2 (E) in a representative experiment of 1 h occlusion 7 days reperfusion. Myofibroblasts in the border zone of a reperfused myocardial infarct produce SMemb (A). SMemb positive cells are spindle shaped (B,D) and express -SMAc (C), but not SM2 (E), a marker for mature smooth muscle cells. In contrast, the mature smooth muscle cells located in the media of the arteriole (arrowheads), are positive for -SMAc and SM2, but do not express SMemb. (Magnification A:100x, B:400x, C,E,D: 200x).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Myocardial repair involves a superbly orchestrated interaction of cells, cytokines, growth factors and extracellular matrix proteins [8,14–17,33,34]. Coronary artery occlusion is associated with cardiomyocyte death due to necrosis or apoptosis and triggers an intense inflammatory reaction. One of the early features of the inflammatory response is accumulation of neutrophils, chemotactically attracted by C5a and IL-8 [34,35]. The main function of the neutrophils appears to be the removal and degradation of dead myocytes and tissue debris. Granulocyte infiltration is followed by chemotactic attraction of monocytes and lymphocytes, predominantly mediated through induction of Transforming Growth Factor-β (TGF-β) and Monocyte Chemotactic Protein-1 (MCP-1) [36]. Following 72 h of reperfusion mast cells also accumulate in the reperfused myocardium [10] and may serve as an important source of fibrogenic substances [37]. Macrophages and mast cells provide a rich source of cytokines and growth factors necessary to support fibroblast proliferation and neovessel formation.

4.1 Circumflex coronary artery occlusion in the dog–a model of acute myocardial infarction
In the current study, we investigated the phenotypic characteristics of myofibroblasts in healing myocardial scars, utilizing a canine model of experimental myocardial infarction [10,14,24,25]. Occlusion of the circumflex coronary artery, followed by reperfusion, causes a relatively small area of necrosis and gives us the opportunity to study a "pure" model of ischemic cardiac injury and repair, without the potential influence of hemodynamic compromise due to infarction of a large territory. Although microembolization has a role in the pathophysiology of acute coronary syndromes [38], myocardial infarction in man is in most cases secondary to acute thrombotic occlusion of a coronary vessel [39], a situation very similar to our experimental model.

4.2 Cell proliferation in the healing myocardial scar
During the early stages of healing the infarcted heart is a cell rich tissue, containing a variety of inflammatory cells, fibroblasts and endothelial cells. Although chemotactic attraction of circulating blood cells, or precursors is crucial to the formation of granulation tissue and subsequent repair, proliferation of resident myocardial cells is also important. Using immunolabeling with PCNA [10,40] we identified significant numbers of proliferating cells in the healing myocardium. The PCNA index in ischemic segments significantly increased following 72 h of reperfusion (at 72 h 11.3+1.53% vs. 0.56+0.33% at 5 h; P<0.05, n = 5), and peaked after 5 to 7 days of reperfusion reflecting the high proliferative activity in the healing scar. After 3 to 4 weeks the PCNA index decreased (4.8+1.1% at 28 days vs. 13.7+2.25% at 5 days; P<0.05, n = 5), marking the transition to the maturation phase of the scar, characterized by a lower proliferative activity.

4.3 Microvessel density following myocardial ischemia and reperfusion
Neovascularization of the healing myocardium is crucial to cardiac repair. Formation of blood vessels allows delivery of oxygen and nutrients to the cells participating in the healing process. The angiogenic process relies on capillary sprouting from preexisting blood vessels, as well as on the migration and incorporation of bone marrow derived endothelial progenitor cells [41]. The mediators responsible for the vessel formation have not been fully elucidated, however, the contribution of a wide variety of cytokines and growth factors has been suggested [42–44]. Our current study describes the dynamic changes in microvessel density during maturation of the healing myocardial scar and suggests continuous neovessel formation for at least three weeks following myocardial ischemia and reperfusion. We report a significant increase in microvessel density in fibrotic areas when compared with non-fibrotic areas after 21 days of reperfusion (fibrotic areas: 3808.5+443.5 microvessels/sq mm vs. non fibrotic areas: 2066+107.9 microvessels/sq mm; P<0.05, n = 5). Our findings are consistent with those reported by Arras and coworkers [45] in a porcine model of cardiac microembolization suggesting significant capillary growth starting 3 to 7 days after embolization, that decreased after 4 weeks.

4.4 Phenotypic modulation of myofibroblasts during healing
We have previously identified (10) the majority of proliferating cells in the healing canine heart as -SMAc positive myofibroblasts. Willems and colleagues [17] have characterized these interstitial nonvascular spindle shaped cells, which were present in human myocardial scars 4 to 6 days after an infarction. Further studies suggested that myofibroblasts are the predominant source of collagen mRNA in healing myocardial infarcts [46]. Myofibroblasts transiently appear during granulation tissue formation [47] and become apoptotic when the scar matures [48]. TGF-β appears to have an important role in myofibroblast differentiation during wound healing by regulating -SMAc expression in these cells [49]. Persistent expression of -SMAc by fibroblasts has been described for at least 8 weeks after a nonreperfused myocardial infarct in the rat [18]. Our experiments demonstrated the presence of nonvascular -SMAc positive cells in the healing heart after 72 h of reperfusion (Fig. 4). -SMAc expression peaked after 5 to 14 days of reperfusion and decreased after 21 to 28 days, when the scar became less cellular (Fig. 3). The myofibroblasts were positive for vimentin, but did not express smooth muscle myosin, calponin and desmin (Fig. 4,5).

4.5 Myofibroblasts express SMemb and not SM2
Smooth muscle myosin heavy chains exist in multiple isoforms [50–53]. Smooth muscle cells may contain at least three types of myosin heavy chain isoforms: SM1, SM2 and SMemb. SM1 and SM2 are specific markers for smooth muscle cells, whereas SMemb is a nonmuscle-type myosin heavy chain abundantly expressed in embryonal and dedifferentiated smooth muscle cells. SM2, in particular, is a useful marker for mature, differentiated smooth muscle cells [28–30,50–53]. Our experiments demonstrated that, much like in human heart, medial vascular myocytes in adult canine epicardial coronary arteries, but not coronary arterioles, express SMemb. Furthermore, the majority of nonvascular -SMAc positive cells in the border zone of reperfused canine myocardial infarcts expressed SMemb, but not SM2, suggesting an embryonal phenotype (Fig. 6). These cells are phenotypically modulated myofibroblasts. In contrast, arteriolar vascular myocytes in control and infarcted areas were positive for SM2 and negative for SMemb. These cells are mature smooth muscle cells.

4.6 Role of SMemb expressing myofibroblasts in the healing scar
We present the first demonstration of myofibroblasts expressing SMemb in healing myocardial scars. The role of SMemb expression in wound healing remains unclear. Recent experiments [54] have shown that myofibroblasts in the healing scar express a homologue of the Drosophila tissue polarity gene frizzled (fz2), when migrating into the granulation tissue, which may be involved in the spatial control of cardiac wound repair after infarction. Interestingly, Kuro-o and coworkers [55] suggested that dedifferentiation of smooth muscle cells and expression of SMemb is coupled with smooth muscle cell proliferation. In addition SMemb expressing myofibroblast-like cells were recently identified in rejected cardiac allografts [56]. Other studies [57] indicated that SMemb positive smooth muscle cells in abdominal aortic aneurysms may show increased metalloproteinase expression suggesting that phenotypic modulation may be associated with an enhanced role of these cells in extracellular matrix metabolism.

SMemb expression may reflect the dedifferentiation and phenotypic plasticity of myofibroblasts following cardiac injury, which may facilitate wound repair. Myofibroblasts are undifferentiated cells capable of evolving towards a particular phenotype depending on the microenvironmental factors and the specific physiologic needs [20–22]. Thus, myofibroblasts are responsible for extracellular matrix deposition in the healing scar [46]. They may also serve as precursor cells necessary for angiogenesis.

Expression of smooth muscle markers, such as -SMAc and SMemb by myofibroblasts may be important in mediating wound contraction during cardiac repair. Furthermore, -SMAc is a signal for retardation of migratory behavior in fibroblasts [58]. Thus, its expression during healing of a myocardial infarct may serve to immobilize them in areas of injury and to prevent uncontrolled fibrosis and infarct expansion.

The mechanism responsible for SMemb expression remains unclear. Recently, Watanabe and coworkers [59] demonstrated that Basic Transcription regulatory Element Binding Protein 2 (BTEB2) mediates the transcriptional regulation of SMemb in vascular smooth muscle cells. In addition, BTEB2 mRNA expression by smooth muscle cells is rapidly and persistently upregulated by basic Fibroblast Growth Factor in vitro [60]. The factors responsible for phenotypic modulation and SMemb induction in myofibroblasts infiltrating the healing scar have not been investigated, however growth factors, such as TGF-β and bFGF may have a significant role.

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Healing of the infarcted myocardium is a dynamic process associated with influx of several different cell types crucial in cardiac repair. Our studies demonstrated active proliferation [10] and increased numbers of -SMAc positive myofibroblasts during the proliferative phase of scar formation (3–14 days). These cells also showed expression of the embryonal isoform of smooth muscle myosin heavy chain (SMemb). Induction of dedifferentiation markers, such as SMemb, underlines the phenotypic plasticity of the myofibroblast population, which includes cells that may be capable of assuming a variety of different roles, such as extracellular matrix metabolism, neovessel formation and contractile activity. Furthermore, the increased expression of -SMAc may be responsible for retardation of the migratory behavior in myofibroblasts. Phenotypic modulation is crucial for wound formation, as it provides the healing myocardium with a large population of cells with a high proliferative potential and phenotypic plasticity, which is confined to the injured area. Understanding of the characteristics of myofibroblasts in healing myocardial infarcts is important in order to design therapeutic interventions aimed at improving post-infarct cardiac recovery.

Time for primary review 27 days.

	Acknowledgements

 
This work was supported by NIH Grant HL-42550 and the DeBakey Heart Center. We wish to thank Alida Evans, Peggy Jackson and Stephanie Butcher for expert technical assistance and Concepcion Mata and Sharon Malinowski for editorial assistance with the manuscript.

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Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 

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