- Email: [email protected]
Differential Expression of Transforming Growth Factor-β1 Is Associated With Fetal Regeneration After Myocardial Infarction
Maggie M. Hodges, MD, Carlos Zgheib, PhD, Junwang Xu, PhD, Junyi Hu, MD, Lindel C. Dewberry, MD, Sarah A. Hilton, MD, Myron W. Allukian, MD, Joseph H. Gorman, III, MD, Robert C. Gorman, MD, and Kenneth W. Liechty, MD Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, Colorado; Department of Pediatric Surgery, The University of Texas Health Science Center at Houston, Houston, Texas; and Department of Surgery and Gorman Cardiovascular Research Group, University of Pennsylvania, Philadelphia, Pennsylvania
Background. Global extracellular matrix (ECM)-related gene expression is decreased after myocardial infarction (MI) in fetal sheep when compared with adult sheep. Transforming growth factor (TGF)-b1 is a key regulator of ECM; therefore we hypothesize that TGF-b1 is differentially expressed in adult and fetal infarcts after MI. Methods. Adult and fetal sheep underwent MI via ligation of the left anterior descending coronary artery. Expression of TGF-b1 and ECM-related genes was evaluated by ovine-specific microarray and quantitative polymerase chain reaction. Fibroblasts from the left ventricle of adult and fetal hearts were treated with TGF-b1 or a TGF-b1 receptor inhibitor (LY36497) to evaluate the effect of TGF-b1 on ECM-related genes. Results. Col1a1, col3a1, and MMP9 expression were increased in adult infarcts 3 and 30 days after MI but were upregulated in fetal infarcts only 3 days after MI. Three days after MI elastin expression was increased in
adult infarcts. Despite upregulation in adult infarcts both 3 and 30 days after MI, TGF-b1 was not upregulated in fetal infarcts at any time point. Inhibition of the TGF-b1 receptor in adult cardiac fibroblasts decreased expression of col1a1, col3a1, MMP9, elastin, and TIMP1, whereas treatment of fetal cardiac fibroblasts with TGF-b1 increased expression of these genes. Conclusions. TGF-b1 is increased in adult infarcts compared with regenerative, fetal infarcts after MI. Although treatment of fetal cardiac fibroblasts with TGFb1 conveys an adult phenotype, inhibition of TGF-b1 conveys a fetal phenotype to adult cardiac fibroblasts. Decreasing TGF-b1 after MI may facilitate myocardial regeneration by “fetalizing” the otherwise fibrotic, adult response to MI.
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matrix metalloproteinases (MMPs), leading to degradation of the extracellular matrix (ECM) and facilitating migration of inflammatory cells into the infarct [6]. The proliferative phase is accompanied by cardiomyocyte recruitment and activation of fibroblasts to the profibrotic myofibroblast phenotype [6–8]. Finally during the maturation phase collagen is cross-linked, producing a stiff, collagen-rich scar [8, 9]. These responses result in adverse ventricular remodeling, driving the impaired ventricular function defining HF. Our large mammalian model of myocardial regeneration after MI has shown that mid-gestation fetal sheep regenerate fully functional myocardium 30 days after MI [10, 11]. Unsurprisingly the fetal response to MI is strikingly different from the adult response. Adult myocardium responds to MI with early, persistent upregulation
eart disease has become the leading cause of death worldwide [1]. In the United States, ischemic heart disease is responsible for over 800,000 deaths per year, with an economic impact exceeding $32 billion per year [2, 3]. Despite the decreasing incidence of acute myocardial infarction (MI), heart failure (HF) prevalence has increased 12.5%, with 25% of patients developing clinical signs and symptoms of HF after MI [2, 4]. Despite advances in HF management, the 5-year mortality after an HF diagnosis remains 54%, underscoring the need to develop effective therapies to prevent progression from MI to HF [5]. After MI the heart progresses through sequential but overlapping phases of inflammation, proliferation, and remodeling. During the inflammatory phase reactive oxygen species and cytokines stimulate the synthesis of
Accepted for publication Dec 17, 2018. Address correspondence to Dr Hodges, The Laboratory for Fetal and Regenerative Biology, 12631 E 17th Ave, C302, Aurora, CO 80045; email: [email protected].
Ó 2019 by The Society of Thoracic Surgeons Published by Elsevier Inc.
(Ann Thorac Surg 2019;108:59–66) Ó 2019 by The Society of Thoracic Surgeons
The Supplemental Material can be viewed in the online version of this article [https://doi.org/10.1016/j.athoracsur. 2018.12.042] on http://www.annalsthoracicsurgery.org.
0003-4975/$36.00 https://doi.org/10.1016/j.athoracsur.2018.12.042
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of genes related to wounding, inflammation, cell proliferation and migration, and ECM remodeling [12]. The fetal response to MI is characterized by a muted upregulation of these same pathways, with return to baseline gene expression accompanying myocardial regeneration 30 days after MI [12]. Modulating the fibrotic response after MI is a target of therapies directed at alleviating the morbidity and mortality of HF [13–15]. Modest improvement in clinical outcomes is attributed to therapies that indirectly moderate fibrosis, specifically by decreasing the activity of the ECM regulator transforming growth factor (TGF)-b1 [6, 16]. Stimulated by mechanical strain, the effect of MMPs, disruption of the ECM, and activation of angiotensin signaling, TGF-b1 is believed to be one of the key initiators of the transition from the inflammatory phase to the proliferative phase of wound healing [17]. In addition to playing a role in this transition, TGF-b1 activates cardiac fibroblasts to the pro-fibrotic myofibroblast phenotype, leading to increased collagen I and collagen III and contributing to fibrosis of the infarct area post-MI [9]. We have previously demonstrated reduced global ECMrelated gene expression in the fetal response, both 3 and 30 days post-MI; however the role of TGF-b1 in the fetal response to MI has yet to be examined in a large mammalian model. Given the known regulation of ECM by TGF-b1 we hypothesize that TGF-b1 is differentially expressed in adult and fetal infarcts after MI.
Material and Methods All experiments were approved by The University of Colorado Denver Institutional Animal Care and Use Committee and The University of Pennsylvania Institutional Animal Care and Use Committee and were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996), and the European Convention on Animal Care. Please see Supplemental Methods for further details.
Results Histology Three days post-MI, hematoxylin and eosin staining of adult and fetal hearts demonstrated hemorrhage, loss of tissue architecture, and presence of a basophilic, inflammatory infiltrate (Fig 1). Thirty days post-MI, hematoxylin and eosin staining demonstrated continued loss of tissue architecture in adult hearts, with abundant ECM deposition. Fetal hearts 30 days post-MI were characterized by restoration of tissue architecture. Three days post-MI, adult and fetal infarcts demonstrated scant collagen deposition, as seen using Masson’s trichrome stain (Fig 2). However, 30 days post-MI adult infarcts demonstrated widespread collagen deposition, with negligible collagen deposition observed in fetal infarcts (Fig 2). These histologic findings correspond to our previously reported functional outcomes, in which fetal hearts demonstrate
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preservation of ejection fraction 3 and 30 days after MI, whereas adult hearts demonstrate decreased ejection fraction 3 days after MI, with a 17% decrease in ejection fraction 30 days after MI [10].
Ovine-Specific Microarray The ovine-specific microarray included 74 of 141 distinct ECM-related genes identified by the Gene Ontology Consortium database. Twenty-two ECM-related genes were unaltered in all samples and were not further evaluated in our analysis. Three days post-MI fetal hearts demonstrated no significant difference in expression of col1a1 or col3a1. However adult hearts demonstrated significant upregulation of both col1a1 (18.17-fold) and col3a1 (10.22-fold) 3 days post-MI, as shown in Supplemental Figure 1. The abundant collagen deposition in adult infarcts 30 days post-MI was associated with upregulation of col1a1 (15.75-fold) and col3a1 (11.46-fold). Elastin was significantly decreased in adult infarcts 3 days post-MI (1.96-fold decrease) and upregulated 30 days post-MI (10.49-fold increase). MMP9 was significantly upregulated in fetal infarcts 30 days postMI (2.45-fold). Three days post-MI, MMP9 was significantly upregulated in adult infarcts (9.02-fold) and remained upregulated 30 days post-MI, although this upregulation did not reach statistical significance (5.65fold increase, p value for the statistical analysis of microarrays ¼ 0.06). TIMP1 was not altered in fetal infarcts post-MI; however microarray results suggest that TIMP1 was significantly upregulated in adult infarcts 3 days (31.72-fold) and 30 days (10.06-fold) post-MI. These results are depicted in Figure 3 and detailed in Supplemental Figure 1. Literature review identified tumor necrosis factor (TNF)a, pTEN, TGF-b1, and TGF-b3 as known modulators of ECM [18–22]. TNF-a was significantly upregulated in both fetal (2.56-fold) and adult (4.14-fold) infarcts 3 days postMI. Thirty days post-MI TNF-a gene expression returned to baseline in fetal infarcts, whereas expression in adult infarcts remained significantly upregulated (4.56-fold). pTEN expression was unchanged in fetal infarcts post-MI; however pTEN expression was significantly upregulated in adult infarcts 30 days post-MI (2.21-fold). TGF-b1 was not significantly altered in fetal infarcts post-MI; however TGF-b1 was significantly upregulated (2.84-fold) in adult infarcts 3 days post-MI, with a nonsignificant elevation by 30 days post-MI. Finally although TGF-b3 was modestly upregulated in fetal infarcts 30 days post-MI (1.45-fold), TGF-b3 was significantly increased in adult infarcts both 3 and 30 days post-MI (5.59-fold and 3.68-fold, respectively). Network analysis revealed that most significantly upregulated reactomes were related to ECM expression. Specifically the largest nodes in our network analysis were related to TP53 (cell cycle regulation, apoptosis), FLNA (filamin A, actin binding protein), and TGF-b1 (Fig 3).
Quantitative Polymerase Chain Reaction Validation of the microarray was performed using quantitative polymerase chain reaction, with results depicted in Figures 4 and 5. Fetal infarcts demonstrated
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Fig 1. Infarct subsections were stained with hematoxylin and eosin after fixation in 4% paraformaldehyde (original magnification 10). Representative subsections of fetal (A) 3 day, (B) adult 3 day, (C) fetal 30 day, and (D) adult 30 day infarct areas are presented. Scale bars ¼ 100 mm.
modest upregulation of col1a1 3 days post-MI, with return to baseline by day 30. Conversely adult infarcts demonstrated continued upregulation of col1a1 3 and 30 days post-MI, as shown in Figure 4. Similarly, col3a1 was upregulated in fetal infarcts 3 days after MI, with return to baseline 30 days after MI. However col3a1 remained significantly upregulated in adult infarcts both 3 days and
30 days post-MI. Elastin was not significantly altered either 3 or 30 days post-MI in fetal infarcts; however elastin was significantly upregulated in adult infarcts both 3 days and 30 days post-MI. MMP9 was upregulated in fetal infarcts 3 days post-MI, with return to baseline by 30 days post-MI. In contrast MMP9 was robustly upregulated in adult infarcts both 3 and 30 days post-MI. TIMP1
Fig 2. Infarct subsections were stained with Masson’s trichrome after fixation in 4% paraformaldehyde (original magnification 10). Representative subsections of (A) fetal 3 day, (B) adult 3 day, (C) fetal 30 day, and (D) adult 30 day infarct areas are presented. Scale bars ¼ 100 mm.
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Fig 3. Network analysis depicting the relationship and relative expression of genes identified by the gene ontology term “extracellular matrix” in adult hearts 3 days after myocardial infarction. Genes identified as key components and regulators of extracellular matrix gene expression are identified by colored circles, and color corresponds to the fold change in infarct area gene expression compared with remote zone gene expression.
was significantly decreased in adult and fetal infarcts, both 3 and 30 days post-MI. Three days post-MI quantitative polymerase chain reaction confirmed that fetal infarcts are associated with upregulation of TNF-a, pTEN, and TGF-b3. Consistent with regeneration of infarcted fetal myocardium the expression of pTEN, TGF-b1, and TGF-b3 in fetal infarcts 30 days post-MI was not significantly different from baseline. Interestingly, TNF-a was significantly reduced in fetal infarcts 30 days post-MI. Adult infarcts demonstrated significant upregulation of pTEN, TGF-b1, and TGF-b3 3 days post-MI, without significant upregulation of TNF-a. In association with the increased fibrosis observed histologically the adult infarcts demonstrated persistent upregulation of TNF-a, pTEN, TGF-b1, and TGF-b3 30 days post-MI (Fig 5).
Effect of TGF-b1 on Cardiac Fibroblasts Adult cardiac fibroblasts demonstrated significant upregulation of col1a1, col3a1, elastin, MMP9, and TIMP1 gene expression when compared with fetal cardiac fibroblasts (Fig 6). Given microarray results suggesting a significant role for TGF-b1 in the regulation of ECM-related gene expression after MI, adult and fetal cardiac fibroblasts were treated with either LY36497 (a TGF-b1 receptor inhibitor) or recombinant TGF-b1 for 24 hours. Treatment of adult cardiac fibroblasts with LY36497 reduced
expression of col1a1, col3a1, elastin, and MMP9 while increasing expression of TIMP1. Conversely treatment of the fetal cardiac fibroblasts with TGF-b1 increased the expression of col1a1, col3a1, elastin, MMP9, and TIMP1, conveying an adult phenotype to fetal fibroblasts.
Comment We have shown significantly reduced global ECM-related gene expression in the fetal response to MI compared with the adult response [23]. We are now the first to demonstrate that fetal myocardial regeneration is associated with an absence of TGF-b1 upregulation. These results suggest that fetal myocardial regeneration may in part be the result of decreased TGF-b1 expression after MI. Furthermore the role of TGF-b1 is underscored by demonstrating that fetal cardiac fibroblasts treated with TGF-b1 assume an adult phenotype, whereas inhibition of TGF-b1 “fetalizes” the gene expression profile of adult cardiac fibroblasts. TGF-b1 has been evaluated as a potential therapeutic target in the treatment of HF. In vivo studies have produced discrepant results regarding the impact of TGF-b1 on ventricular remodeling post-MI; however the pleiotropic effect of TGF-b1 on inflammatory and fibrotic pathways is likely responsible for these results [6, 24].
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Fig 4. Quantitative polymerase chain reaction evaluation of extracellular matrix–related gene expression. (A) Col1a1, (B) Col3a1, (C) Elastin, (D) MMP9, and (E) TIMP1 gene expression was evaluated in adult and fetal hearts, 3 and 30 days after myocardial infarction. Target gene expression is normalized to expression of the housekeeping gene GAPDH, and infarct area (IA) gene expression is normalized to remote zone (RZ) gene expression. (D3 ¼ day 3; D30 ¼ day 30.)
TGF-b1 is known to activate cardiac fibroblasts to the pro-inflammatory, pro-fibrotic myofibroblast phenotype, which is characterized by the increased production of col1a1, col3a1, and MMP9 via canonical and non-canonical
TGF-b1 signaling pathways, as show in Figure 7 [25]. Absence of TGF-b1 upregulation in fetal infarcts was associated with attenuated collagen deposition, attenuated col1a1, col3a1, and MMP9 gene expression and an Fig 5. Quantitative polymerase chain reaction validation of microarray results focusing on known regulators of extracellular matrix–related gene expression. (A) Tumor necrosis factor (TNF)-a, (B) pTEN, (C) transforming growth factor (TGF)-b1, and (D) TGF-b3 gene expression was evaluated in adult and fetal hearts, 3 and 30 days after myocardial infarction. Target gene expression is normalized to expression of the housekeeping gene GAPDH, and infarct area (IA) gene expression is normalized to remote zone (RZ) gene expression. (D3 ¼ day 3; D30 ¼ day 30.)
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Fig 6. Quantitative polymerase chain reaction evaluation of extracellular matrix–related gene expression in adult and fetal cardiac fibroblasts. (A) Col1a1, (B) Col3a1, (C) Elastin, (D) MMP9, and (E) TIMP1 gene expression was evaluated in adult and fetal cardiac fibroblasts at baseline, in fetal cardiac fibroblasts treated with 10 ng/mL transforming growth factor (TGF)-b1 for 24 hours, and in adult cardiac fibroblasts treated with a TGF-b1 inhibitor (TGF-b1i) for 24 hours.
attenuated decrease in TIMP1 gene expression in our model. Furthermore inhibition of TGFb1 in adult cardiac fibroblasts decreased expression of col1a1, col3a1, elastin, and MMP9 and increased expression of TIMP1, a gene expression profile more consistent with that of fetal cardiac fibroblasts. Given the differential expression of TGFb1 in adult and fetal infarcts, these results suggest that decreased expression of TGF-b1 in fetal infarcts may play a key role in fetal myocardial regeneration after MI. Collagen I and collagen III are the principal collagens found in cardiac muscle and are believed responsible for cardiac fibrosis after MI. Our results confirm the positive association between col1a1 and col3a1 gene expression and collagen deposition reported previously [26, 27] and demonstrate an attenuated expression of col1a1 and col3a1 in fetal hearts, suggesting that the regenerative, fetal response to MI is characterized by a reduced fibrotic response as early as 3 days post-MI. This reduced fibrotic response may enable the migration of pro-regenerative macrophages and cardiac progenitor cells to the infarct, potentiating the regenerative response we have observed in fetal hearts after MI [11, 23]. Elastin, a key structural protein found in the healing myocardium, is believed to preserve the structural
integrity of the infarcted myocardium, conveying increased strength and elasticity to an otherwise rigid and fibrotic scar [28]. Our results failed to demonstrate a change in elastin gene expression in the fetal, regenerative response to MI. Conversely adult infarcts demonstrated early and persistent upregulation of elastin expression, similar to the pattern of observed TGF-b1 gene expression. Fetal cardiac fibroblasts treated with TGF-b1 demonstrated increased elastin expression, whereas adult cardiac fibroblasts treated with a TGF-b1 receptor inhibitor demonstrated reduced elastin expression. This suggests that differential elastin expression in adult and fetal hearts may be related to differential expression of TGF-b1 post-MI. MMP9 is a potent collagenase and gelatinase whose upregulation increases migration of cardiac fibroblasts [29]. Interestingly, an in vivo model using MMP9-/- mice observed improved mortality and decreased left ventricular dilation in mice lacking functional MMP9, suggesting that MMP9 upregulation may be pathologic after MI [30]. These seemingly disparate results indicate that MMP9 may be responsible for early, beneficial effects and pathologic, late effects post-MI. We observed significant upregulation of MMP9 (14.15-fold increase) 3 days post-MI in fetal
Fig 7. Depiction of classic, SMAD dependent “canonical” versus non–SMAD dependent “noncanonical” pathways of transforming growth factor (TGF)-b1 activation. Latent TGF-b1, bound in the extracellular matrix (ECM), is released in response to numerous factors, including mechanical strain, the breakdown of ECM by matrix metalloproteinases (MMPs), and the presence of reactive oxygen species (ROS) and matricellular proteins such as thrombospondin-1 (TSP-1) [25]. Activation of these pathways leads to increased expression of ECM-related genes.
hearts, with return to baseline by 30 days post-MI. The early upregulation of MMP9 may enable the migration of cardiac progenitor cells and pro-angiogenesis/proremodeling macrophages to the infarct area, facilitating the regeneration of functional myocardium observed in our fetal model. The striking upregulation of MMP9 (168fold) that we observed in adult hearts 3-days post-MI suggests an over-abundance of MMP9 may drive the balance of ECM proteolysis from a state of modest “relaxation,” where proteolysis facilitates cytokine-directed cellular migration into the infarct, to a state dominated by frank degradation, in which lysis of collagen cross-linkages leads to ventricular dilation and loss of systolic function. TIMP1 is a ubiquitous, multifunctional protein that inhibits most MMPs, including MMP9, while also playing a role in cell growth and angiogenesis [31]. TIMP1 gene expression was decreased in adult and fetal samples at all time points. However TIMP1 gene expression was more markedly reduced in adult infarcts 3 days post-MI (3.35fold decrease vs 1.8-fold decrease in fetal infarcts). Conversely MMP9 is upregulated in fetal infarcts (14.2fold increase) and adult infarcts (168-fold increase) at the same time point. The more marked upregulation of MMP9, in conjunction with the more pronounced decrease in TIMP1, is consistent with an imbalanced proteolytic milieu that may contribute to ventricular dilation observed in adult hearts after MI. Furthermore
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treatment of adult cardiac fibroblasts with an inhibitor of TGF-b1 resulted in an increase in TIMP1 gene expression, suggesting that decreasing TGF-b1 activity after MI may improve ventricular function after MI by restoring the TIMP1/MMP9 balance. TGF-b1 is a potent, pleiotropic regulator of ECM production and fibroblast phenotype in numerous disease models; our results suggest that TGF-b1 plays a similar, integral role in the development of HF after MI [32, 33]. However our results are limited by the scope of the current study. The chosen time points may limit evaluation of ECM regulation during the inflammatory phase of wound healing, precluding us from commenting on the fragile balance of ECM breakdown and synthesis necessary to preserve myocardial structure immediately after MI [17]. However the previously reported inflammatory cellular infiltrate and increase in global inflammationrelated gene expression in both fetal and adult infarcts 3 days post-MI suggests our results capture a glimpse of the late inflammatory phase [10, 12]. Future evaluation of additional time points will enable a detailed analysis of the role TGF-b1 plays in ECM deposition responsible for preservation of ventricular structure after MI, in comparison with the role of TGF-b1 during ECM deposition during the proliferative and remodeling phases of wound healing. Although limited success has been gained by targeting TGF-b1 signaling in the heart, small molecule therapies are increasingly used with successful targeting of TGF-b1 in other disease models [34, 35]. The efficacy of these therapies in ameliorating the development of HF after MI has yet to be examined. In conclusion, the dual impact of an aging global population and a rising burden of chronic disease is leading to a growing prevalence of risk factors for cardiovascular disease and a growing incidence of HF [36, 37]. Without the development of effective therapies aimed at mitigating this growing burden of disease, the morbidity associated with HF will continue to increase on a global scale. We have contrasted the expression and regulation of ECM-related genes in the fibrotic, adult response to MI to the regenerative, fetal response to MI. Absence of TGF-b1 upregulation is associated with regeneration of fully functional myocardium in fetal hearts. Furthermore, adult cardiac fibroblasts can be “fetalized” via inhibition of TGF-b1. Further studies are warranted to fully evaluate TGF-b1 as a therapeutic target in the effort to relieve the growing global burden of disease attributable to HF after MI.
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Background. Global extracellular matrix (ECM)-related gene expression is decreased after myocardial infarction (MI) in fetal sheep when compared with adult sheep. Transforming growth factor (TGF)-b1 is a key regulator of ECM; therefore we hypothesize that TGF-b1 is differentially expressed in adult and fetal infarcts after MI. Methods. Adult and fetal sheep underwent MI via ligation of the left anterior descending coronary artery. Expression of TGF-b1 and ECM-related genes was evaluated by ovine-specific microarray and quantitative polymerase chain reaction. Fibroblasts from the left ventricle of adult and fetal hearts were treated with TGF-b1 or a TGF-b1 receptor inhibitor (LY36497) to evaluate the effect of TGF-b1 on ECM-related genes. Results. Col1a1, col3a1, and MMP9 expression were increased in adult infarcts 3 and 30 days after MI but were upregulated in fetal infarcts only 3 days after MI. Three days after MI elastin expression was increased in
adult infarcts. Despite upregulation in adult infarcts both 3 and 30 days after MI, TGF-b1 was not upregulated in fetal infarcts at any time point. Inhibition of the TGF-b1 receptor in adult cardiac fibroblasts decreased expression of col1a1, col3a1, MMP9, elastin, and TIMP1, whereas treatment of fetal cardiac fibroblasts with TGF-b1 increased expression of these genes. Conclusions. TGF-b1 is increased in adult infarcts compared with regenerative, fetal infarcts after MI. Although treatment of fetal cardiac fibroblasts with TGFb1 conveys an adult phenotype, inhibition of TGF-b1 conveys a fetal phenotype to adult cardiac fibroblasts. Decreasing TGF-b1 after MI may facilitate myocardial regeneration by “fetalizing” the otherwise fibrotic, adult response to MI.
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matrix metalloproteinases (MMPs), leading to degradation of the extracellular matrix (ECM) and facilitating migration of inflammatory cells into the infarct [6]. The proliferative phase is accompanied by cardiomyocyte recruitment and activation of fibroblasts to the profibrotic myofibroblast phenotype [6–8]. Finally during the maturation phase collagen is cross-linked, producing a stiff, collagen-rich scar [8, 9]. These responses result in adverse ventricular remodeling, driving the impaired ventricular function defining HF. Our large mammalian model of myocardial regeneration after MI has shown that mid-gestation fetal sheep regenerate fully functional myocardium 30 days after MI [10, 11]. Unsurprisingly the fetal response to MI is strikingly different from the adult response. Adult myocardium responds to MI with early, persistent upregulation
eart disease has become the leading cause of death worldwide [1]. In the United States, ischemic heart disease is responsible for over 800,000 deaths per year, with an economic impact exceeding $32 billion per year [2, 3]. Despite the decreasing incidence of acute myocardial infarction (MI), heart failure (HF) prevalence has increased 12.5%, with 25% of patients developing clinical signs and symptoms of HF after MI [2, 4]. Despite advances in HF management, the 5-year mortality after an HF diagnosis remains 54%, underscoring the need to develop effective therapies to prevent progression from MI to HF [5]. After MI the heart progresses through sequential but overlapping phases of inflammation, proliferation, and remodeling. During the inflammatory phase reactive oxygen species and cytokines stimulate the synthesis of
Accepted for publication Dec 17, 2018. Address correspondence to Dr Hodges, The Laboratory for Fetal and Regenerative Biology, 12631 E 17th Ave, C302, Aurora, CO 80045; email: [email protected].
Ó 2019 by The Society of Thoracic Surgeons Published by Elsevier Inc.
(Ann Thorac Surg 2019;108:59–66) Ó 2019 by The Society of Thoracic Surgeons
The Supplemental Material can be viewed in the online version of this article [https://doi.org/10.1016/j.athoracsur. 2018.12.042] on http://www.annalsthoracicsurgery.org.
0003-4975/$36.00 https://doi.org/10.1016/j.athoracsur.2018.12.042
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of genes related to wounding, inflammation, cell proliferation and migration, and ECM remodeling [12]. The fetal response to MI is characterized by a muted upregulation of these same pathways, with return to baseline gene expression accompanying myocardial regeneration 30 days after MI [12]. Modulating the fibrotic response after MI is a target of therapies directed at alleviating the morbidity and mortality of HF [13–15]. Modest improvement in clinical outcomes is attributed to therapies that indirectly moderate fibrosis, specifically by decreasing the activity of the ECM regulator transforming growth factor (TGF)-b1 [6, 16]. Stimulated by mechanical strain, the effect of MMPs, disruption of the ECM, and activation of angiotensin signaling, TGF-b1 is believed to be one of the key initiators of the transition from the inflammatory phase to the proliferative phase of wound healing [17]. In addition to playing a role in this transition, TGF-b1 activates cardiac fibroblasts to the pro-fibrotic myofibroblast phenotype, leading to increased collagen I and collagen III and contributing to fibrosis of the infarct area post-MI [9]. We have previously demonstrated reduced global ECMrelated gene expression in the fetal response, both 3 and 30 days post-MI; however the role of TGF-b1 in the fetal response to MI has yet to be examined in a large mammalian model. Given the known regulation of ECM by TGF-b1 we hypothesize that TGF-b1 is differentially expressed in adult and fetal infarcts after MI.
Material and Methods All experiments were approved by The University of Colorado Denver Institutional Animal Care and Use Committee and The University of Pennsylvania Institutional Animal Care and Use Committee and were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996), and the European Convention on Animal Care. Please see Supplemental Methods for further details.
Results Histology Three days post-MI, hematoxylin and eosin staining of adult and fetal hearts demonstrated hemorrhage, loss of tissue architecture, and presence of a basophilic, inflammatory infiltrate (Fig 1). Thirty days post-MI, hematoxylin and eosin staining demonstrated continued loss of tissue architecture in adult hearts, with abundant ECM deposition. Fetal hearts 30 days post-MI were characterized by restoration of tissue architecture. Three days post-MI, adult and fetal infarcts demonstrated scant collagen deposition, as seen using Masson’s trichrome stain (Fig 2). However, 30 days post-MI adult infarcts demonstrated widespread collagen deposition, with negligible collagen deposition observed in fetal infarcts (Fig 2). These histologic findings correspond to our previously reported functional outcomes, in which fetal hearts demonstrate
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preservation of ejection fraction 3 and 30 days after MI, whereas adult hearts demonstrate decreased ejection fraction 3 days after MI, with a 17% decrease in ejection fraction 30 days after MI [10].
Ovine-Specific Microarray The ovine-specific microarray included 74 of 141 distinct ECM-related genes identified by the Gene Ontology Consortium database. Twenty-two ECM-related genes were unaltered in all samples and were not further evaluated in our analysis. Three days post-MI fetal hearts demonstrated no significant difference in expression of col1a1 or col3a1. However adult hearts demonstrated significant upregulation of both col1a1 (18.17-fold) and col3a1 (10.22-fold) 3 days post-MI, as shown in Supplemental Figure 1. The abundant collagen deposition in adult infarcts 30 days post-MI was associated with upregulation of col1a1 (15.75-fold) and col3a1 (11.46-fold). Elastin was significantly decreased in adult infarcts 3 days post-MI (1.96-fold decrease) and upregulated 30 days post-MI (10.49-fold increase). MMP9 was significantly upregulated in fetal infarcts 30 days postMI (2.45-fold). Three days post-MI, MMP9 was significantly upregulated in adult infarcts (9.02-fold) and remained upregulated 30 days post-MI, although this upregulation did not reach statistical significance (5.65fold increase, p value for the statistical analysis of microarrays ¼ 0.06). TIMP1 was not altered in fetal infarcts post-MI; however microarray results suggest that TIMP1 was significantly upregulated in adult infarcts 3 days (31.72-fold) and 30 days (10.06-fold) post-MI. These results are depicted in Figure 3 and detailed in Supplemental Figure 1. Literature review identified tumor necrosis factor (TNF)a, pTEN, TGF-b1, and TGF-b3 as known modulators of ECM [18–22]. TNF-a was significantly upregulated in both fetal (2.56-fold) and adult (4.14-fold) infarcts 3 days postMI. Thirty days post-MI TNF-a gene expression returned to baseline in fetal infarcts, whereas expression in adult infarcts remained significantly upregulated (4.56-fold). pTEN expression was unchanged in fetal infarcts post-MI; however pTEN expression was significantly upregulated in adult infarcts 30 days post-MI (2.21-fold). TGF-b1 was not significantly altered in fetal infarcts post-MI; however TGF-b1 was significantly upregulated (2.84-fold) in adult infarcts 3 days post-MI, with a nonsignificant elevation by 30 days post-MI. Finally although TGF-b3 was modestly upregulated in fetal infarcts 30 days post-MI (1.45-fold), TGF-b3 was significantly increased in adult infarcts both 3 and 30 days post-MI (5.59-fold and 3.68-fold, respectively). Network analysis revealed that most significantly upregulated reactomes were related to ECM expression. Specifically the largest nodes in our network analysis were related to TP53 (cell cycle regulation, apoptosis), FLNA (filamin A, actin binding protein), and TGF-b1 (Fig 3).
Quantitative Polymerase Chain Reaction Validation of the microarray was performed using quantitative polymerase chain reaction, with results depicted in Figures 4 and 5. Fetal infarcts demonstrated
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Fig 1. Infarct subsections were stained with hematoxylin and eosin after fixation in 4% paraformaldehyde (original magnification 10). Representative subsections of fetal (A) 3 day, (B) adult 3 day, (C) fetal 30 day, and (D) adult 30 day infarct areas are presented. Scale bars ¼ 100 mm.
modest upregulation of col1a1 3 days post-MI, with return to baseline by day 30. Conversely adult infarcts demonstrated continued upregulation of col1a1 3 and 30 days post-MI, as shown in Figure 4. Similarly, col3a1 was upregulated in fetal infarcts 3 days after MI, with return to baseline 30 days after MI. However col3a1 remained significantly upregulated in adult infarcts both 3 days and
30 days post-MI. Elastin was not significantly altered either 3 or 30 days post-MI in fetal infarcts; however elastin was significantly upregulated in adult infarcts both 3 days and 30 days post-MI. MMP9 was upregulated in fetal infarcts 3 days post-MI, with return to baseline by 30 days post-MI. In contrast MMP9 was robustly upregulated in adult infarcts both 3 and 30 days post-MI. TIMP1
Fig 2. Infarct subsections were stained with Masson’s trichrome after fixation in 4% paraformaldehyde (original magnification 10). Representative subsections of (A) fetal 3 day, (B) adult 3 day, (C) fetal 30 day, and (D) adult 30 day infarct areas are presented. Scale bars ¼ 100 mm.
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Fig 3. Network analysis depicting the relationship and relative expression of genes identified by the gene ontology term “extracellular matrix” in adult hearts 3 days after myocardial infarction. Genes identified as key components and regulators of extracellular matrix gene expression are identified by colored circles, and color corresponds to the fold change in infarct area gene expression compared with remote zone gene expression.
was significantly decreased in adult and fetal infarcts, both 3 and 30 days post-MI. Three days post-MI quantitative polymerase chain reaction confirmed that fetal infarcts are associated with upregulation of TNF-a, pTEN, and TGF-b3. Consistent with regeneration of infarcted fetal myocardium the expression of pTEN, TGF-b1, and TGF-b3 in fetal infarcts 30 days post-MI was not significantly different from baseline. Interestingly, TNF-a was significantly reduced in fetal infarcts 30 days post-MI. Adult infarcts demonstrated significant upregulation of pTEN, TGF-b1, and TGF-b3 3 days post-MI, without significant upregulation of TNF-a. In association with the increased fibrosis observed histologically the adult infarcts demonstrated persistent upregulation of TNF-a, pTEN, TGF-b1, and TGF-b3 30 days post-MI (Fig 5).
Effect of TGF-b1 on Cardiac Fibroblasts Adult cardiac fibroblasts demonstrated significant upregulation of col1a1, col3a1, elastin, MMP9, and TIMP1 gene expression when compared with fetal cardiac fibroblasts (Fig 6). Given microarray results suggesting a significant role for TGF-b1 in the regulation of ECM-related gene expression after MI, adult and fetal cardiac fibroblasts were treated with either LY36497 (a TGF-b1 receptor inhibitor) or recombinant TGF-b1 for 24 hours. Treatment of adult cardiac fibroblasts with LY36497 reduced
expression of col1a1, col3a1, elastin, and MMP9 while increasing expression of TIMP1. Conversely treatment of the fetal cardiac fibroblasts with TGF-b1 increased the expression of col1a1, col3a1, elastin, MMP9, and TIMP1, conveying an adult phenotype to fetal fibroblasts.
Comment We have shown significantly reduced global ECM-related gene expression in the fetal response to MI compared with the adult response [23]. We are now the first to demonstrate that fetal myocardial regeneration is associated with an absence of TGF-b1 upregulation. These results suggest that fetal myocardial regeneration may in part be the result of decreased TGF-b1 expression after MI. Furthermore the role of TGF-b1 is underscored by demonstrating that fetal cardiac fibroblasts treated with TGF-b1 assume an adult phenotype, whereas inhibition of TGF-b1 “fetalizes” the gene expression profile of adult cardiac fibroblasts. TGF-b1 has been evaluated as a potential therapeutic target in the treatment of HF. In vivo studies have produced discrepant results regarding the impact of TGF-b1 on ventricular remodeling post-MI; however the pleiotropic effect of TGF-b1 on inflammatory and fibrotic pathways is likely responsible for these results [6, 24].
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Fig 4. Quantitative polymerase chain reaction evaluation of extracellular matrix–related gene expression. (A) Col1a1, (B) Col3a1, (C) Elastin, (D) MMP9, and (E) TIMP1 gene expression was evaluated in adult and fetal hearts, 3 and 30 days after myocardial infarction. Target gene expression is normalized to expression of the housekeeping gene GAPDH, and infarct area (IA) gene expression is normalized to remote zone (RZ) gene expression. (D3 ¼ day 3; D30 ¼ day 30.)
TGF-b1 is known to activate cardiac fibroblasts to the pro-inflammatory, pro-fibrotic myofibroblast phenotype, which is characterized by the increased production of col1a1, col3a1, and MMP9 via canonical and non-canonical
TGF-b1 signaling pathways, as show in Figure 7 [25]. Absence of TGF-b1 upregulation in fetal infarcts was associated with attenuated collagen deposition, attenuated col1a1, col3a1, and MMP9 gene expression and an Fig 5. Quantitative polymerase chain reaction validation of microarray results focusing on known regulators of extracellular matrix–related gene expression. (A) Tumor necrosis factor (TNF)-a, (B) pTEN, (C) transforming growth factor (TGF)-b1, and (D) TGF-b3 gene expression was evaluated in adult and fetal hearts, 3 and 30 days after myocardial infarction. Target gene expression is normalized to expression of the housekeeping gene GAPDH, and infarct area (IA) gene expression is normalized to remote zone (RZ) gene expression. (D3 ¼ day 3; D30 ¼ day 30.)
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Fig 6. Quantitative polymerase chain reaction evaluation of extracellular matrix–related gene expression in adult and fetal cardiac fibroblasts. (A) Col1a1, (B) Col3a1, (C) Elastin, (D) MMP9, and (E) TIMP1 gene expression was evaluated in adult and fetal cardiac fibroblasts at baseline, in fetal cardiac fibroblasts treated with 10 ng/mL transforming growth factor (TGF)-b1 for 24 hours, and in adult cardiac fibroblasts treated with a TGF-b1 inhibitor (TGF-b1i) for 24 hours.
attenuated decrease in TIMP1 gene expression in our model. Furthermore inhibition of TGFb1 in adult cardiac fibroblasts decreased expression of col1a1, col3a1, elastin, and MMP9 and increased expression of TIMP1, a gene expression profile more consistent with that of fetal cardiac fibroblasts. Given the differential expression of TGFb1 in adult and fetal infarcts, these results suggest that decreased expression of TGF-b1 in fetal infarcts may play a key role in fetal myocardial regeneration after MI. Collagen I and collagen III are the principal collagens found in cardiac muscle and are believed responsible for cardiac fibrosis after MI. Our results confirm the positive association between col1a1 and col3a1 gene expression and collagen deposition reported previously [26, 27] and demonstrate an attenuated expression of col1a1 and col3a1 in fetal hearts, suggesting that the regenerative, fetal response to MI is characterized by a reduced fibrotic response as early as 3 days post-MI. This reduced fibrotic response may enable the migration of pro-regenerative macrophages and cardiac progenitor cells to the infarct, potentiating the regenerative response we have observed in fetal hearts after MI [11, 23]. Elastin, a key structural protein found in the healing myocardium, is believed to preserve the structural
integrity of the infarcted myocardium, conveying increased strength and elasticity to an otherwise rigid and fibrotic scar [28]. Our results failed to demonstrate a change in elastin gene expression in the fetal, regenerative response to MI. Conversely adult infarcts demonstrated early and persistent upregulation of elastin expression, similar to the pattern of observed TGF-b1 gene expression. Fetal cardiac fibroblasts treated with TGF-b1 demonstrated increased elastin expression, whereas adult cardiac fibroblasts treated with a TGF-b1 receptor inhibitor demonstrated reduced elastin expression. This suggests that differential elastin expression in adult and fetal hearts may be related to differential expression of TGF-b1 post-MI. MMP9 is a potent collagenase and gelatinase whose upregulation increases migration of cardiac fibroblasts [29]. Interestingly, an in vivo model using MMP9-/- mice observed improved mortality and decreased left ventricular dilation in mice lacking functional MMP9, suggesting that MMP9 upregulation may be pathologic after MI [30]. These seemingly disparate results indicate that MMP9 may be responsible for early, beneficial effects and pathologic, late effects post-MI. We observed significant upregulation of MMP9 (14.15-fold increase) 3 days post-MI in fetal
Fig 7. Depiction of classic, SMAD dependent “canonical” versus non–SMAD dependent “noncanonical” pathways of transforming growth factor (TGF)-b1 activation. Latent TGF-b1, bound in the extracellular matrix (ECM), is released in response to numerous factors, including mechanical strain, the breakdown of ECM by matrix metalloproteinases (MMPs), and the presence of reactive oxygen species (ROS) and matricellular proteins such as thrombospondin-1 (TSP-1) [25]. Activation of these pathways leads to increased expression of ECM-related genes.
hearts, with return to baseline by 30 days post-MI. The early upregulation of MMP9 may enable the migration of cardiac progenitor cells and pro-angiogenesis/proremodeling macrophages to the infarct area, facilitating the regeneration of functional myocardium observed in our fetal model. The striking upregulation of MMP9 (168fold) that we observed in adult hearts 3-days post-MI suggests an over-abundance of MMP9 may drive the balance of ECM proteolysis from a state of modest “relaxation,” where proteolysis facilitates cytokine-directed cellular migration into the infarct, to a state dominated by frank degradation, in which lysis of collagen cross-linkages leads to ventricular dilation and loss of systolic function. TIMP1 is a ubiquitous, multifunctional protein that inhibits most MMPs, including MMP9, while also playing a role in cell growth and angiogenesis [31]. TIMP1 gene expression was decreased in adult and fetal samples at all time points. However TIMP1 gene expression was more markedly reduced in adult infarcts 3 days post-MI (3.35fold decrease vs 1.8-fold decrease in fetal infarcts). Conversely MMP9 is upregulated in fetal infarcts (14.2fold increase) and adult infarcts (168-fold increase) at the same time point. The more marked upregulation of MMP9, in conjunction with the more pronounced decrease in TIMP1, is consistent with an imbalanced proteolytic milieu that may contribute to ventricular dilation observed in adult hearts after MI. Furthermore
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treatment of adult cardiac fibroblasts with an inhibitor of TGF-b1 resulted in an increase in TIMP1 gene expression, suggesting that decreasing TGF-b1 activity after MI may improve ventricular function after MI by restoring the TIMP1/MMP9 balance. TGF-b1 is a potent, pleiotropic regulator of ECM production and fibroblast phenotype in numerous disease models; our results suggest that TGF-b1 plays a similar, integral role in the development of HF after MI [32, 33]. However our results are limited by the scope of the current study. The chosen time points may limit evaluation of ECM regulation during the inflammatory phase of wound healing, precluding us from commenting on the fragile balance of ECM breakdown and synthesis necessary to preserve myocardial structure immediately after MI [17]. However the previously reported inflammatory cellular infiltrate and increase in global inflammationrelated gene expression in both fetal and adult infarcts 3 days post-MI suggests our results capture a glimpse of the late inflammatory phase [10, 12]. Future evaluation of additional time points will enable a detailed analysis of the role TGF-b1 plays in ECM deposition responsible for preservation of ventricular structure after MI, in comparison with the role of TGF-b1 during ECM deposition during the proliferative and remodeling phases of wound healing. Although limited success has been gained by targeting TGF-b1 signaling in the heart, small molecule therapies are increasingly used with successful targeting of TGF-b1 in other disease models [34, 35]. The efficacy of these therapies in ameliorating the development of HF after MI has yet to be examined. In conclusion, the dual impact of an aging global population and a rising burden of chronic disease is leading to a growing prevalence of risk factors for cardiovascular disease and a growing incidence of HF [36, 37]. Without the development of effective therapies aimed at mitigating this growing burden of disease, the morbidity associated with HF will continue to increase on a global scale. We have contrasted the expression and regulation of ECM-related genes in the fibrotic, adult response to MI to the regenerative, fetal response to MI. Absence of TGF-b1 upregulation is associated with regeneration of fully functional myocardium in fetal hearts. Furthermore, adult cardiac fibroblasts can be “fetalized” via inhibition of TGF-b1. Further studies are warranted to fully evaluate TGF-b1 as a therapeutic target in the effort to relieve the growing global burden of disease attributable to HF after MI.
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