A total of 2528 articles were identified using database searching, and 2212 were recorded after duplicates removal. One thousand seven hundred thirty (1730) were excluded after screening of title/abstract, 215 were finally excluded (because when many separate articles were present with similar conclusions, only those were selected to be included which mainly focused on TF Isl1, Brg1/Baf60 – Smarcd3 complex, Nkx2-5, GATA4, Tbx5, Mef2c, HAND1/2, MYOCD, MSX2, HOPX, wnt-signaling pathway, Notch, FGF, BMPs for their roles in cardiogenesis/cardiomyocyte development, proliferation, differentiation, tumor suppression, tumorigenesis and cardiac myxoma, and 3 articles were excluded during data extraction. Finally, 264 articles were included.
The limitations of this study are mentioned in the ‘methodology’, ‘study design’, and other respective sections. Based on the results of this study, it also further addresses in ‘discussion’ section the findings of this study.
This study traces the factors responsible for the rare occurrence and benign nature of primary cardiac tumors, focusing on the cardiac myxoma, and its relation to the limited cardiac proliferative/ regenerative potential by investigating cardiogenesis through the lens of key cardiac genes/ transcription factors and signaling pathways.
KEY CARDIAC TRANSCRIPTION FACTORS/GENES:
In this section, this study investigates key cardiac transcription factors/genes and their roles in cardiogenesis, progenitor/ proliferation related functions, their interactions with tumor suppressor/ differentiation related genes, contributions to cardiomyocyte specific genetic programing/combinatorial code, their role in other tumors and in the development of cardiac myxoma [26]. This study investigates Isl1, Brg1/Baf60 – Smarcd3 complex, Nkx2-5, GATA4, Tbx5, Mef2c, HAND1/2, MYOCD, MSX2, HOPX [27].
In First Heart Field (FHF), the expression of Nkx2-5 is predominant [28, 29]. In Second Heart Field (SHF), Isl1 expression is of immense significance in multipotent Cardiac Progenitor Cells (CPCs). The CPCs are multi-lineage cells with major expression of Nkx2-5 and Isl1 [30, 31]. By increasing the gene expressions of BMP signaling pathway and downregulating Wnt pathway, the CPCs switch towards cardiomyocyte fate. The Isl1 and Nkx2-5 TFs act via activating cascade of downstream cardiac genes in time specific manner.
It is important to note that Isl1 is a pioneering TF of cardiomyocyte cell fate. As Isl1 expression begins to decline, the HOPX becomes upregulated. The Nkx2-5 has very strong interactions with HOPX. The HOPX is expressed in cardiomyoblast and is very important in the process of differentiation as it is also expressed in pre-cardiac mesoderm. HOPX positively interacts with BMPs, SMADs and negatively with Wnt-signaling and Axin2 signaling pathway. The Wnt-pathway and Axin2 oppose differentiation of CPCs [32, 33].
In CPCs, the Nkx2-5 expression continuously increases over the duration of differentiation. When HOPX is defective, Wnt-pathway becomes upregulated and this downregulates Nkx2-5. Normally, the BMP-SMAD complex is activated by HOPX. This downregulates WNT-signaling pathway and promotes differentiation of CPCs in cardiomyocyte. This BMP-SMAD complex increases MSX1 expression to promote differentiation and downregulates Axin2 [34].
Role in cardiogenesis:
Islet1 (Isl1) plays the role of a pioneering transcription factor in epigenetic control of cardiomyocyte cell fate [35]. This also governs epigenetic programming and shapes chromatin landscape. It works with additional regulatory factors to specify cell lineage and cardiac differentiation [36]. The Isl1 shapes genetic landscape of cardiac progenitor cells (CPCs). This governs a regulatory network of genes that is involved in unfolding cardiac lineage [37]. There is so much still unknown about Islet1. It is transiently expressed in SHF progenitor cells including atrial area. It is involved in proliferation, survival and migration of SHF CPCs [38]. When Islet1 is defective, cardiac development gets disrupted. Isl1 expression is more profound before SHF progenitor cells differentiate into heart tube [39].
Progenitor or Proliferation-related roles:
Pioneering TF such as Isl1 is not sufficient for gene activation on its own but imparts competence for transcription. This leads to gene chromatin remodeling. With additional TFs and additional regulatory proteins, it induces cardiac cell type specific gene expression which contributes to lineage specification and differentiation of cardiomyocytes. The Isl1 is one of the earliest genes expressed in the cardiac progenitors. Isl1 interacts strongly with Tbx1 and both are strongly expressed in multipotent CPCs. It also has major interaction with Wnt-signaling pathway. Isl1 also works with FGF10 to promote proliferation of progenitor cells [40, 41, 42]. Isl1 expression plays important roles also in the neural crest cells, and in heart the progenitor cells expressing Isl1 are capable of differentiating into cardiomyocytes, endothelial and smooth muscle lineage. The CPCs also maintain expression of Isl1 in postnatal state. The Islet1 expression is essential in renewal of cardiac progenitors. Prior to differentiation, the gene expression of Islet1 contributes to proliferation of CPCs [43, 44].
Key Interactions with tumor suppressors/ differentiation-related genes:
It sets in motion the gene expression of Brg1- Baf60 which contributes to commit CPCs towards cardiomyocyte fate. Nkx2-5 acts as a repressor of Isl1 and downregulates its gene expression for the purpose of differentiation [45]. GATA4 activates Isl1 enhancer. The BMPs downregulate the gene expression of Isl1 and Tbx1 to promote myocardial differentiation [46].
Contributions to combinatorial code/ cell type specific genetic-programming:
The induction of cell type specific genetic-programming needs pioneering TFs such as Isl1 which works with other special TFs in the form of combinations specific to cell type. This combinatorial code determines the cell type and regulates its progression towards cell fate specific differentiated state [47, 48].
Presence in other tumors:
Upregulated expression of Islet1 is present in many cancers including pheochromocytoma, pancreatic, gastrointestinal, lung tumors, bile duct carcinoma, prostate and breast cancers. Islet1 expression is also present in insulinoma cells, bladder cancer, NHL, glioma, melanoma and others [49, 50, 51, 52, 53]. Isl1 is a novel regulator of cyclins and c-myc gene. This also signifies the role of Isl1 in tumor development [54].
Possible role in cardiac myxoma:
Despite being very similar to multipotent cardiac progenitor cells, cardiac myxomas are c-kit positive but very rarely Isl1 positive [55]. As the Nkx2-5 has been found to be involved in cardiac myxoma development, hence its role in interacting with Isl1 is very significant. Possibly, the influence of Nkx2-5 over the genetic landscape in cardiac myxoma prevents the progression of cardiac myxoma cells towards malignant state. The combinatorial effect of all differentiation-related genes and tumor suppressors on the genetic landscape is possibly a key factor that may serve to limit cardiac regenerative potential but also makes the occurrence of primary cardiac tumors a rare phenomenon [56, 57].
The Isl1 and GATA4 also interact with Estrogen Receptor alpha. This may be a contributing factor in the development of more cardiac myxoma cases in female patients [58, 59, 60].
- Brg1/Baf60 – Smarcd3 complex:
Role in cardiogenesis:
It is massively expressed in anterior/SHF [61]. Its defects exhibit cardiac morphogenetic defects as it acts as transcriptional regulator. It promotes progenitors towards cardiomyocyte fate. Its over-expression has been found to accelerate the activation of cardiac lineage related target genes [62, 63].
Progenitor or Proliferation-related roles:
When this complex is under the influence of wnt-signaling, it contributes to epithelial-mesenchymal transition (EMT). The Baf60 is capable of inducing proliferation in progenitor cells, but with SMARCD3 complex its function becomes so much different and it contributes to cardiac differentiation. In neural progenitors, it interacts with notch to promote proliferation [64, 65].
Key Interactions with tumor suppressors/ differentiation-related genes:
It mediates interactions with core cardiac TFs including Tbx5, Nkx2-5 and GATA4. It promotes binding of GATA4 and Tbx5 to cardiac specific genes. This induces downstream regulatory networks. The process of cardiac differentiation becomes defective when there are defects in this complex [66]. SMARCD3 is also considered to play tumor suppressor role [67, 68].
Contributions to combinatorial code/ cell type specific genetic-programming:
This complex when combined with GATA4 and Tbx5, is capable of switching on the cardiac gene expression in non-cardiac regions. It is capable of driving mesenchymal cells towards cardiac fate. This complex has major interactions with GATA4 to turn on cell type specific cardiac gene expression. Then it combines with Tbx5 to repress the gene expression of non-cardiac genes. They have massive control over cardiac differentiation and may have fundamental influence over cardiac regeneration potential. This complex is one of the key parts of cardiac specific cell programming [69, 70].
Presence in other tumors:
It plays multiple roles in different cancers depending on the microenvironment and also based on the influence of predominant signaling pathways such as Wnt, TGF- beta and MAPK. In colorectal cancer, it works with Wnt-pathway to promote metastasis. But in breast cancer, it plays the role as a possible tumor suppressor [71, 72].
Possible role in cardiac myxoma:
There is not much data about the role of this complex in cardiac myxoma. This may be because of the fact that there are not as many studies on cardiac myoma as much are present on colorectal cancer or glioblastoma or on other major tumors. But by investigating the role of this complex in the process of cardiogenesis, this study explains the possible ways this genetic complex may be playing significant role in this tumor development and in defining the regenerative potential of cardiomyocytes. The role that this gene complex plays is variable and is also dependent on the gene expression of other key regulatory genes/TFs and signaling pathways. Such as with Wnt-signaling, it promotes proliferation of progenitor cells, and with GATA4 and Tbx5 it promotes the differentiation process. Similarly, in cardiac myxoma development the expression of Baf60 complex may become dysregulated and serve variable role depending on the landscape of cardiac myxoma at specific moment. [1,7, 19]
- Nkx2-5:
Role in cardiogenesis: The TF Nkx2-5 is among the very first cardiac specific patterning genes. The site of its expression is called heart forming region and plays key roles in cardiac specification, differentiation and proliferation. The significance of Nkx2-5 can be estimated from the fact that defects in Nkx2-5 are involved in congenital heart defects as well. It decides atrial and ventricular fate. It is one of the four key regulators of cardiac cell type. It is expressed in precursor cardiac cells and leads to proper cardiac development [73, 74].
Progenitor or Proliferation-related roles:
This gene is of key significance as it results in cascade of downstream signaling and induces the cardiac programming in pluripotent mesenchymal stem cells. It is dependent on JAK-STAT pathway. The TF Nkx2-5 controls cardiomyocyte differentiation by working with Mef2c which is a key enhancer of Nkx2-5 [75]. The most significant aspect of this TF Nkx2-5 that may be very important in terms of investigating cardiac myxoma etiology, it is that Nkx2-5 is first expressed in cardiac progenitor cells and its gene expression is downregulated temporarily during the differentiation phase of cardiomyocytes. But a constant low level of Nkx2-5 gene expression persists throughout life. It is involved in the induction of initial phases but not in late phases of cardiomyocyte development. It interacts with notch signaling pathway to promote proliferation of cardiac progenitor cells. The fate determination of cardiovascular lineages is regulated by Nkx2-5 in the earliest specified multipotent cardiac progenitors. Early multipotent cardiovascular progenitor cells expressing Nkx2-5 give rise to endothelial lineages, smooth muscles cells and cardiomyocytes [76, 77, 78].
Key Interactions with tumor suppressors/ differentiation-related genes:
It has major interactions with GATA4 and Tbx5. It establishes a positive feedback loop with GATA4. It directs cardiac looping by working with MEF2c, Hand1 and Hand2. Working with Nkx2-7, the TF Nkx2-5 is involved in maintenance of cardiomyocyte cellular identity. The expression of Nkx2-5 is so much significant during the process of differentiation of cardiomyocytes as it acts as a repressor of FGF10 and Isl1, to promote differentiation. Nkx2-5 promotes cardiac phenotype by antagonizing TBX1 which promotes proliferation in SHF progenitor cells [79, 80, 81]. It interacts with BMP signaling pathway for the purpose of differentiation. As Nkx2-5 is cardiac specific patterning TF, its expression represses non-cardiac genes while inducing the gene expression of cardiogenic genes. The presence of Nkx2-5 expression sets controls in cardiac cells that give them cell type specific features such as the permanent nature of cardiac cell type. TF Nkx2-5 working as master regulator of cardiac development influences and induces a cascade of regulatory and developmental genes. This sets in motion the cell type specific combinatorial code that governs cardiomyogenesis. In post-natal cardiomyocytes, it is needed for proper functioning. NPCEDRG is a novel tumor suppressive gene and has potential binding sites for NKX2-5 and this way it is capable of inducing cell differentiation, control cell growth and regulate the cell cycle [82]. Nkx2-5 influences and regulates gene activity of HOPX to modulate cardiac gene expression. HOPX has a potential tumor suppressive activity [83].
Contributions to combinatorial code/ cell type specific genetic-programming:
Nkx2-5 sets in motion a cascade of combinatorial genetic interactions that govern the genetic programming of cardiogenesis. The Nkx2-5 based early patterning sets stage for BMP and Notch based gene expression. The defects in the Nkx2-5 down-regulate the BMPs and Notch signaling pathway, resulting in the disruption of cardiogenesis [84]. TF Nkx2-5 auto-regulates itself and is further mainly regulated by GATA4 and SMAD proteins. Its expression is also dependent on Islet1. In the induction of TF Nkx2-5, expression of BMP2/4 is required and the activity of Wnt-pathway is inhibited. The Wnt-signaling negatively impacts the TF Nkx2-5 gene expression, hence must be inhibited. The BMPs induces the FGF8 signaling to promote development of cardiac proteins. TF Nkx2-5 is involved in the upregulation of HAND1 and HAND2, resulting in the differentiation and proliferation of cardiac cells. The TF Nkx2-5 also has major interactions with p53. It also interacts with FGF16 and FGF10. Through its interactions, it controls proliferation and differentiation [85, 86, 87].
Presence in other tumors:
The role of Nkx2-5 is different in different microenvironments. It can promote both proliferation and differentiation in different situations [88]. Nkx2-5 is dysregulated in ALL, HCC, T cell neoplasias, methylated in prostate adenocarcinoma, hyper-methylated in salivary gland adenoid cystic carcinoma, oncogenic mef2c in ALL is principally deregulated via activating leukemic transcription factor Nkx2-5, notch3 signaling in leukemic T cells is activated by Nkx2-5, dysregulated Nkx2-5 expression in sarcomas, hypermethylated in breast, prostate and colon cancer [89, 90]. It is expressed in PTC and reduces the expression of thyroid differentiating markers. There is age related Nkx2-5 methylation in normal prostate tissues and may predispose to prostate adenocarcinoma [91, 92, 93]. In ALL, Nkx2-5 has direct interactions with GATA genes and MEF2C oncogenic expression is influenced by Nkx2-5. The MEF2C expression inhibits apoptosis promoting NR4A1/NUR77 expression. Nkx2-5 is not expressed in hematopoietic stem cells, but in ALL it contributes to oncogenesis and interacts with BCL11 [94, 95, 96]. It is important to note that TF Nkx2-5 deletions cause thyroid hypoplasia and this signifies its role in survival, proliferation and different roles in different microenvironments.
Possible role in cardiac myxoma:
Some studies have hinted towards the possible role of TF Nkx2-5 in this tumor development [97]. Nkx2.5/Csx, GATA-4, MEF2, and eHAND are key involved genes in cardiac myxomas. Defects in Nkx2-5 cause abnormalities in atrial growth and development [98]. The Nkx2-5, Oct-4, Isl1, c-kit are upregulated and this produces cardiac progenitor stem cell-like state.
This study postulates that deviation of cardiomyocyes from cell type specific well-differentiated state results in turning back of the cells into progenitor-like cardiac stem cells. The hallmark of this process is the upregulation of Nkx2-5 gene expression. TF Nkx2-5 has major interactions with p53 tumor suppressor gene that also prevents this tumor from becoming malignant. Cardiomyocytes have very limited proliferation potential in adult life. This nature of cardiomyocytes is governed by the cell type specific programming that also restricts the proliferative potential of this cell type after completion of cardiogenesis. The Nkx2-5 exerts vast control over proliferation. It has been found that it has wide range of functions depending on where it is being expressed as it enhances the gene expression of Mesenchymal Stem Cells (MSCs) in transplant patients and controls cardiac progenitor cell proliferations [99, 100].
The heterogeneity that exists in cardiac myxoma, its existence may be because of the multitude of roles that Nkx2-5 and other key genes/TFs and signaling pathways play in different microenvironments and in different cell types. The dysregulations in them result in the deviation of cells away from cell-type specific gene expression. As the cardiac specific combinatorial code based functioning of TFs get dysregulated, this deviates the direction of lineages from one specific to multiple cell types having significant role of these key genes/TFs and signaling pathways in their development.
- GATA4:
Role in cardiogenesis:
GATA4 is a very important regulator of genes in the process of development. It plays a key role in the process of myocardial differentiation. The GATA4 also plays essential role in testicular development. The key interactions include Nkx2-5, TBX5, SRF, HAND2, HDAC2, Erbb3, FOG-1 and FOG-2 [101, 102].
Progenitor or Proliferation-related roles:
GATA4 plays significant role in morphogenesis and promotes cardiomyocyte survival. When GATA4 is deleted or defective, Erb and Erk expression is down-regulated. They both normally play key role in EMT. It down-regulates the c-myc gene expression to promote differentiation process in cardiomyocytes during the process of development. It also regulates hypertrophic growth of heart. Although GATA4 interacts with p53 and p21, but it also works with Bcl2. This GATA4-Bcl2 interaction promotes cardiomyocyte survival [103, 104].
Key Interactions with tumor suppressors/ differentiation-related genes:.
It is expressed in both embryonic and adult cardiomyocytes. It regulates the gene expression of many downstream cardiac genes. It maintains the cardiac function in adult heart. GATA4 is an important regulator of terminal differentiation program in cardiomyocytes. It antagonizes c-myc to limit the replication potential. Multiple studies have suggested that damage to GATA4 also damages the TBX5. This damage also contributes to congenital heart defects [105]. GATA4 plays a very significant role in differentiation process also by governing genes associated with cell-to-cell adhesion, cytoskeleton organization and extracellular matrix dynamics. And this promotes them to become more differentiated and less proliferative [106]. It interacts with p53 and p21, which have tumor suppressor effects. It is important to note that GATA4 interacts with CD40L and this way GATA4 is capable of inducing senescence. GATA4 also acts as a switch to activate NF‑κB signaling [107].
Contributions to combinatorial code/ cell type specific genetic-programming:
It works with other key cardiac TFs including Nkx2-5 and Tbx5. The GATA4 is considered to be a key regulator of cardiac phenotype. It has upstream interactions with BMP, FGF and WNT signaling pathways [108]. The significance of GATA4 can also be estimated from the fact that when ectopically its expression is induced together with TBX5 and SMARCD3, this is capable of inducing genetic programming of cardiomyogenesis in non-cardiac regions of embryo. GATA4 regulates Mef2c expression and acts also as Isl1 enhancer. Here it is important to note that GATA4 which primarily is involved in differentiation of cardiomyocytes, it is interacting with Mef2c and Isl1 both of which are involved in regulating progenitor and proliferation related genes in CPCs [109]. Both GATA4-TBX5 and MEF2C-TBX5 work in triggering the gene expression of subsequent downstream cardiomyocyte specific genes. The GATA4 and TBX5 are considered key regulators of cardiac gene regulatory networks. Nkx2-5 – GATA4 complex also plays role in cardiac hypertrophy in response to stretch. This complex interaction also governs the release of ANP and BNP [110, 111].
Presence in other tumors:
In lung cancer, it plays the role of tumor suppressor as it down-regulates the wnt7b and TGF-beta. The presence of SMAD4 and GATA4 is considered to be related to poor-prognosis in esophageal adenocarcinoma. Similarly, GATA4 is also upregulated in pancreatic cancer and different models have showed that the purpose of its upregulation is to increase the process of differentiation [112, 113]. The presence of GATA4 in different cancers seems to occur for the purpose of upregulating the process of differentiation but in different tumor microenvironments, it fails to halt or reduce proliferation. It may be because of the same reason that in hepatoblastomas, the GATA4 is upregulated in mesenchymal malignant like cells while has a negligible gene expression in normal hepatocytes. Its upregulation in ovarian tumors may also be because of same reasons [114, 115]. In ALL, GATA4 has been associated with increased proliferation and inhibition of apoptosis. This tumor promoting role of GATA4 is influenced by tumor specific interactions and tumor microenvironment. The predominant effect of specific genes and signaling pathways that are governing the landscape of a tumor may undermine the specific function of many differentiation related genes [116, 117].
Possible role in cardiac myxoma:
Primitive cardiomyocyte TFs have been detected in cardiac myxoma including GATA4, Mef2c, Nkx2-5 and eHAND [118]. They are slightly to intensely positive in cardiac myxoma samples. In many samples, GATA4 gene expression was dysregulated. Decline or disruptions in gene expression of key regulatory differentiation genes such as GATA4 may have drastic impact on the overall genetic composition of differentiated cardiomyocytes. Such alterations can disrupt the delicate cell type specific balance of expression among different types of genes/signaling pathways. This may contribute to switch the cells more towards progenitor like state that is hallmark of cardiac myxoma [119, 120].
- Tbx5:
Role in cardiogenesis:
The Tbx5 is one of the key regulators of cardiogenesis. It is involved in promoting differentiation of cardiac progenitor cells into cardiomyocytes. It interacts with NKX2-5, GATA4 and BAF remodeling complex. Studies in which Tbx5 was deleted by CRISPR/Cas9 editing, the cells maintained stem cell like pluripotent state [121, 122]. The Tbx5 is a key player in switching CPCs towards developmental gene expression by inducing differentiation into cardiomyocytes. Mutations in this key TF contribute to Atrial Septal Defect (ASD). It is essential for the development of heart and limbs. It is expressed in embryonic and adult heart. It is also expressed in endocardium of left ventricle [123, 124, 125].
Progenitor or Proliferation-related roles:
In the ventricle, the Tbx5 expression originates from first heart field (FHF) but atrial gene expression originates from Mef2c in second heart field (SHF). Mef2c plays very important role in proliferation of CPCs [126]. Tbx5 works with SHH in the formation of atrial septum. The TF Tbx5 has a very strong relationship with Nkx2-5, and Tbx5 – Nkx2-5 complex contributes to the process of cardiomyocyte differentiation. This complex also prevents activation of non-cardiac genes [127, 128].
Key Interactions with tumor suppressors/ differentiation-related genes:
It is mutated in Holt-Oram syndrome. The Tbx5 promotes other cardiogenic TFs. It is strongly interconnected with GATA4 and damage to GATA4 also damages Tbx5. The TF Tbx5 is so significant for the process of differentiation of cardiomyocytes that when it is defective, it results in apoptosis [129, 130]. Tbx5 interacts with Nkx2-5, GATA4 and BAF60c to drive expression of cardiac genes. Tbx5 also interacts with repressor genes such as NuRD complex, SALL4 and others to downregulate the expression of non-cardiac genes. Tbx5 also induces expression of downstream genes related to cardiomyocyte differentiation including NPPA and GJA5. Just like Tbx5-Nkx2-5 complex, Tbx5 also forms complex with GATA5 and Mef2c to contribute to the process of cardiomyocyte differentiation. These partnerships by Tbx5 play cell type specific key roles in the process of development [131, 132, 133].
Contributions to combinatorial code/ cell type specific genetic-programming:
Tbx5 works with Nkx2-5 to promote cardiac differentiation. The Tbx5 shifts the gene expression profile more towards cardiogenesis and it also plays key role in the beating of cardiomyocytes. In the entire process of cardiac development, the gene expression of Tbx5 is maintained. It also persists in adult heart. The key interactions of Tbx5 include Nkx2-5, GATA4, Baf60c, Mef2c in cardiomyocyte development. It also interacts and regulates the gene expression of a cascade of downstream genes involved in cardiac differentiation. It inhibits the gene expression of neural and other non-cardiac cell types in cardiogenesis through Tbx5-NuRD interaction [134].
Presence in other tumors:
It inhibits cell proliferation in osteosarcoma. It is a critical regulator of oncogenesis. It has been found to suppress the proliferation in NSCLC and acts as a tumor suppressor. Even in normal embryonic developmental processes, its over-expression induces apoptosis and halts cell development. Tbx5 that also acts as tumor suppressor, it is epigenetically inhibited in colorectal cancer [135, 136, 137, 138].
Possible role in cardiac myxoma:
In normal heart, the atrial expression of Tbx5 is far greater than ventricle. And in normal heart, Tbx5-Nkx2-5 forms a complex. This is very important as dysregulated expression of Nkx2-5 is considered to play a very significant role in development of cardiac myxoma. As Tbx5 forms key complexes that have a major effect in cell fate of cardiomyocytes, and Tbx5 is involved in activation and maintenance of cardiac lineage genes. It prevents off-target binding of TFs in cardiac development. Hence, alterations in its gene expression may have profound consequences [139, 140]. It is not expressed in cardiac myxoma. And this may be defining feature in cardiac myxoma development as Tbx5 is one of the principal regulators of cardiomyocyte differentiation. Any dysregulation in Tbx5 can trigger a cascade of destruction by altering the direction of cell type towards mesenchymal progenitor like state. The dysregulations in Tbx5 may be a major contributor in the emergence of heterogeneity in cardiac myxoma because of Tbx5 role in suppression of non-cardiac cell types. [1,7, 19]
- Mef2c:
Role in cardiogenesis, contributions to the combinatorial code/cell type programming and key interactions:
Mef2c works with Nkx2-5 in controlling the differentiation of cardiac progenitor cells (CPCs). GATA4 works also by interacting with both Mef2c and Isl1, and they both have major roles in proliferation of progenitor cells. The different functions of Mef2c are governed by its interactions and the complexes it forms. The Mef2c forms complexes with both key differentiation related genes (GATA4 and Tbx5) of cardiomyocytes. The Mef2c interacts with NF-κB and downregulates its signaling in multiple cell types in endothelial cells. The role of Mef2c is significant because of its individual effect on proliferation and also with the complexes it forms [141, 142]. Mef2c contributes to activation of TF HAND1 [143, 144].
Progenitor or Proliferation-related roles:
Mef2c is involved in cardiac morphogenesis, myogenesis, vascular development and neurogenesis. It contributes to maintaining differentiated state in muscle cells by working with other regulatory complexes. In hematopoiesis, ERK expression proportionally controls Mef2c expression. Mef2c plays oncogenic role in many cancers. One of the very important interactions of Mef2c includes its interactions with Tbx5 and GATA4. The Mef2c also plays key role in the proliferation of cardiac progenitor cells (CPCs) [145, 146].
Presence in other tumors:
Mef2c plays oncogenic role in ALL, AML, colon adenocarcinoma, DLBCL and T-cell lymphomas. It also plays oncogenic role in prostate cancer and interacts with dysregulated notch signaling pathway. In hepatic cancer cells, it increases proliferative signaling. Mef2c acts as an essential transcription factor in AML oncogenesis. It interacts with sox2 during the process of oncogenesis in cancer stem cells [147, 148]. The CDKN1B deletions frequently coincide with expression of MEF2C in ALL. Mef2c also plays oncogenic role in CML and imatinib abrogates its expression. Common cascade pathways (p38 MAPKs-MEF2C) that can result in proliferation, differentiation and apoptosis work with genes IL1R and TGFBR in many breast cancer subtypes. Mef2c and WNT signaling pathway both regulate SIX1 in Hodgkin Lymphoma. Mef2c exerts direct control over Socs2 (a feedback inhibitor of JAK-STAT pathways is expressed in most primitive HSC and is upregulated in response to STAT5-inducing cytokines). The normal response of increased Mef2c expression is upregulation of Socs2. The Mef2c exerts oncogenic effects on Socs2 in different leukemias such as AML and ALL. Mef2c is also upregulated in Rhabdomyosarcomas [149, 150, 151]. Another important role of Mef2c is also seen in pancreatic cancer. YY1 acts as tumor suppressor, suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 which is upregulated by Mef2c.
Possible role in cardiac myxoma:
Multiple studies have detected Mef2c gene expression in cardiac myxoma samples. As Mef2c works in the form of complexes with other key regulatory genes/pathways including GATA4, Isl1, Wnt-pathway, its role is also governed by microenvironment. It is capable of playing oncogenic role. When key differentiation related genes such as GATA4 become dysregulated, this may have drastic impact on the functioning of Mef2c which can ultimately go on to serve like an oncogene in CM landscape. In such conditions, it may switch to work with Wnt and Isl1 resulting in emergence of CPCs like state that is hallmark of cardiac myxoma [152, 153, 154, 155, 156].
- HAND1/2
Role in cardiogenesis, contributions to the combinatorial code/cell type programming and key interactions:
It is expressed in adult heart and is downregulated in cardiomyopathies. It also modulates cardiac hypertrophy. It is also involved in heart, vascular, GIT, limb and neuronal development. The Mef2c contributes to the activation of Hand1. HAND1 plays key role in neural crest development. It also interacts with BMP4 which contributes further to the differentiation of cardiomyocytes [157, 158]. It has major interactions with NKX2-5 and GATA4. It plays role in proliferation with Nkx2-5 and when it interacts with GATA4, it plays role in differentiation of cardiomyocytes. It is important to remember that it also has a tumor suppressor effect [159, 160].
Role in other tumors:
HAND2 also acts as tumor suppressor. It is downregulated in many tumors such as NSCLC and other cancers including ovarian, breast, gastric, colorectal, cervical, endometrial, prostate and esophageal squamous cell cancer [161]. But in micro-environment of hepatocellular carcinoma (HCC) it plays a very different role and promotes tumor development. In normal liver, the gene expression of HAND2 is undetectable. But in some samples of HCC, it has been found downregulated. In HCC, HAND2 interacts with BMP signaling cascade. Due to limitations of data on this role of HAND2, it is not possible to draw concrete conclusions about the role of HAND 2 in HCC [162]. HAND2 negatively regulates TGFbeta, ROCK2 and JAK-STAT pathway [163, 164].
Possible role in cardiac myxoma:
Detected in many but not all cases of cardiac myxoma. It is considered to be involved in the development of cardiac myxoma [165]. The impact and influence of Hand1/2 on the landscape of cardiomyocytes is very important as it also acts as tumor suppressor. Thus, it may have a possible contributing role in limiting the regenerative potential of cardiomyocytes. This may have a contributing role in preventing the malignant transformation and emergence of primary malignant tumors in cardiomyocytes. [1,7, 19]
- MYOCD
Role in cardiogenesis, contributions to the combinatorial code/cell type programming and key interactions:
Mostly MYOCD works with p16 against the TGF-beta signaling, it induces growth arrest and also inhibits cellular proliferation by inhibiting NF-κB signaling. It is important to remember Mef2c also inhibits NF-κB signaling. This is important also because MYOCD-SRF axis forms a major complex with Mef2c to exert control on cardiac progenitors. This is involved in cardiomyocyte survival and maintenance of heart function. When MYOCD is defective, pro-apoptotic factors take over the control of cardiomyocytes. The MYOCD is also involved in maintaining cardiac structural organization [166, 167, 168]. It interacts with Nkx2-5 positively. But when SMAD3 gene expression is present, they interact negatively. The MYOCD also interacts positively with NFAT, HNRNPA1, SRF and Mef2c.
Role in proliferation, differentiation and in some other tumors:
It inhibits stemness in NSCLC as it is an essential tumor suppressor. It is downregulated in lung squamous cell carcinoma and lung adenocarcinoma. It inhibits stemness by inhibiting TGF-beta receptor signaling. The SRF-MYOCD axis is driver of well-differentiated leiomyosarcoma [169]. But MYOCD functions are also governed by the interactive complexes it forms with key regulatory genes. When MYOCD forms an interactive loop with SMAD3/4, it derives TGF-beta based EMT [170, 171]. MYOCD which also has tumor suppressive effect, it is repressed through proliferative signaling by FOXO3A and KLF4/KLF5. The tumor suppressor P53 also has a dose dependent regulatory repressor effect on MYOCD. The GSK3-beta can inhibit MYOCD dependent cardiac gene expression. The activators of MYOCD include p300. The ERK1/2 based phosphorylation of MYOCD inhibits it [172, 173, 174].
Possible role in cardiac myxoma:
There is no proper data on the role of MYOCD in cardiac myxoma. But it may have possible significant role in the process of cardiac tumorigenesis. Based on its loop of interactions and its role in inhibiting the stemness-related progenitor genes and signaling pathways, MYOCD may have profound role in preventing the occurrence of primary tumors in cardiac tissue. As it works positively with Nkx2-5 which is expressed in cardiac myxoma cells, MYOCD may have possible role in maintaining the benign nature of cardiac myxoma and in preventing the occurrence of malignant tumors in cardiac tissue. [1,7, 19]
- MSX2:
Role in cardiogenesis, contributions to the combinatorial code/cell type programming and key interactions:
In cardiogenesis, MSX2 interacts with HAND1/2 and they regulate the gene expression of each other. MSX2 regulates survival of SHF precursors by protecting them against apoptosis. It also makes sure that there is no excessive proliferation of cardiac cells, cardiac neural crest cells and endothelial cells. It acts more as a regulator by interacting with both proliferation related genes and differentiation related genes. MSX1/2 are required for EMT of AV cushions and patterning of atrioventricular myocardium [175, 176, 177].
Role in proliferation, differentiation and in some other tumors:
It functions to maintain a balance between survival and apoptosis. Its upregulation enhances malignant phenotype [178, 179]. It also acts as transcriptional repressor. It induces EMT in pancreatic cancer [180]. MSX2 working with RAS promotes cell growth. MSX2 is downstream target of RAS. The MSX2 expression is upregulated in diabetes and colorectal cancer [181, 182]. Its role in oral SCC is very interesting and significant to understand how it plays multiple roles in different microenvironments. The MSX2 interacts with SOX2 to control cancer stem cell like characterization in oral squamous cell carcinoma. The MSX2 represses tumor stem cell phenotypes by SOX2 dysregulations in oral SCC [183, 184].The in vitro expression of MSX2 has been found to inactivate AKT pathway to promote cell cycle arrest and apoptosis [185].
Possible role in cardiac myxoma:
There is no proper data on the role of MSX2 in cardiac myxoma. As its function is dependent on its interactions and cross-talk, it also varies with microenvironment. Hence, in cardiac myxoma its role is more likely to be state dependent. Such as in advanced cardiac myxoma, it may possibly contribute to tumorigenesis by promoting progenitor-like state. [1,7, 19]
- HOPX:
Role in cardiogenesis, contributions to the combinatorial code/cell type programming and key interactions:
It is expressed in cardio-myoblast intermediate that is committed to cardiomyocyte fate. The niche signals help regulate the committed state. It interacts with activated smads to repress wnt-signaling pathway [186]. It switches the cells more towards differentiated fate of cardiomyocytes by promoting local BMP signals to inhibit wnt-signaling pathway. This results in promoting cardiomyogenesis [187, 188].
Role in proliferation, differentiation and in some other tumors:
HOPX inhibits wnt-signaling, this causes HOPX to trigger stem cell quiescence and this also explains the role of HOPX as tumor suppressor. HOPX plays role of tumor suppressor by acting as RAS inhibitor. The downregulation of HOPX expression contributes to colorectal, head, neck and other cancers. It plays a critical role in cell type homeostasis [189, 190, 191, 192].
Possible role in cardiac myxoma:
There is no proper data on the role of HOPX in cardiac myxoma. The dysregulations in HOPX may possibly serve to contribute towards cardiac myxoma development. The downregulation in its gene expression may alter the genetic landscape of cardiomyocytes as HOPX plays key roles in differentiation and also acts as a tumor suppressor. HOPX dysregulations may lead to switching the gene expression in the direction of progenitor like state as it is present in cardiac progenitor cells (CPCs). [1, 7, 19]
KEY CARDIAC SIGNALING PATHWAYS:
- Wnt signaling pathway
Role in cardiogenesis and key interactions:
It plays a very important role in cardiac development also by contributing to planar cell polarity in cardiogenesis. The Wnt-signaling is also involved in adult heart remodeling. It also contributes to cardiac hypertrophy and increases ANP gene expression. Reduced Wnt levels have been linked to premature myocardial infarction. The wnt3a is involved in cardiac progenitor renewal. This pathway is involved in cardiogenesis and cardiac disease development [193, 194]. Wnt-signaling pathway promotes fibrosis in cardiac repair. This is a very important factor in defining the limitations of cardiac regeneration. The SFRP based downregulation of Wnt/beta-catenin is cardio-protective as it inhibits fibrosis and inflammation. This impact of SFRP gene expression causes endothelial to mesenchymal transition in post myocardial infarction state. The Wnt/beta-catenin pathway promotes proliferation in cardiac progenitor cells and its inhibition promotes differentiation [195, 196].
Role in proliferation, differentiation and in tumorigenesis:
This pathway contributes to stemness in hematopoietic stem cells. In cancers, abnormal wnt-signaling contributes to the maintenance of cancer stem cells. Wnt/beta-catenin is upregulated in ALL and CLL. It interacts with Notch signaling too in cancer microenvironment. The APC tumor suppressor also plays important role in regulating this signaling pathway. Inhibiting Wnt-pathway increases apoptosis in CLL [197, 198, 199]. In melanoma, it promotes tumor growth through abnormal wnt5a. It is also upregulated in breast cancers and its upregulation silences its repressors [200, 201]. The loss of PTEN tumor suppressor and c-myc amplifications are linked to abnormal Wnt-signaling. In tumorigenesis, this pathway derives tumor development [202].
Wnt/beta-catenin pathway has massive influence over other key genes such as tumor suppressors including numb and it is capable of repressing the numb gene expression. This results in the maintenance of cancer stem cells. This is also one of the mechanisms for immune evasion by cancer stem cells. This pathway is also involved in EMT and is upregulated in colorectal cancer, prostate, pancreatic and many other cancers [203, 204].
Possible role in cardiac myxoma:
When Wnt-signaling pathway is disrupted, it contributes to upregulate the gene expression of progenitor like signatures [205]. Wnt/beta-catenin maintains telomeres through TERT gene. When this signaling pathway combines with NF-κB signaling pathway, it contributes to dedifferentiation into stem cell like state [206]. As the Wnt-signaling also plays important role in early stages of cardiogenesis, hence this dedifferentiation related role may have possible implications in cardiac myxoma development.
- FGF Signaling Pathway
Role in cardiogenesis, proliferation and key interactions:
It is involved in the differentiation of stem cells to SHF progenitors and is also involved in the maintenance of pluripotency. These effects are based on interactions and complexes which FGF signaling pathway forms in order to exert effect on cell fate [207]. The FGF2 inhibits TGF-beta1 and promotes cardio-protection. It is also involved in epicardial EMT, coronary vasculogenesis and angiogenesis through FGF1. The FGF Signaling Pathway interacts with the IGF1/2, VEGF, BMPS, TGF-Beta, Wnt and Notch signaling pathway. The FGF10 and FGF8 contribute to proliferation of SHF progenitor cells [208]. The FGF-MAPK axis promotes CPCs multi-potency. The FGFs also have major interaction with PI3K/AKT pathway. In cardiogenesis, FGF2-Wnt complex exerts influence over human pluripotent stem cells to shift them into CPCs by suppressing GSK3-beta [209, 210, 211, 212, 213].
Role in differentiation and in tumorigenesis:
The FGF2-BMP2 complex promotes the cardiomyocyte differentiation. The Isl1-Tbx1 positively interacts with FGF10 which contributes to differentiation of CPCs. The Nkx2-5 negatively regulates FGF10 which is involved in promoting the cardiomyocyte differentiation [214]. In cardiomyocyte differentiation, GATA4 interacts with FGF16 and suppresses proliferation potential. It also provides postnatal cardio-protection. The FGF16 negatively regulates FGF2-RAS-MAPK complex [215, 216].
In postnatal adult cardiomyocytes, FGF Signaling plays very important role in modulating proliferation such as FGF1 is involved in homeostasis and remodeling [217]. FGFs have multifunctional roles ranging from proliferation, homeostasis to differentiation. The FGF acts as blocker of premature CPCs differentiation. The FGF-BMP crosstalk plays key regulatory role in governing cardiomyocyte differentiation [218, 219]. The FGF Signaling Pathway is downregulated by BMP4-MSX1 complex which promotes differentiation of neural crest cells. The FGF Pathway interacts with Nkx2-5 to produce more profoundly the ventricular characteristics in developing heart [220, 221, 222].
Possible role in cardiac myxoma:
The FGF Signaling Pathway may have significant role in cardiac myxoma development as loss of FGF causes gradual accumulation of atrial cells [223, 224, 225]. It is important to note that most cardiac myxomas originate in the atria. The loss of FGF has such immense impact that it causes ectopic atrial gene expression in ventricles. One of the most important impacts of the sustained FGF signaling is that it acts to suppress the cardiomyocyte plasticity. This may also point to the origins of cardiac myxoma.
- BMPs
Role in cardiogenesis and key interactions:
BMPs downregulate the expression of progenitor genes in CPCs. It enhances differentiation of cardiomyocyte. Overall, it induces some progenitor genes as well. The BMP signaling pathway downregulates Isl1, Tbx1, FGF10 and switches the gene expression towards cardiomyocyte differentiation [226]. When BMP-signaling is defective, the gene expression of HAND2 and Nkx2-5 remains unchanged. The BMPs positively regulate Nkx2-5, HAND2, Tbx2 and Tbx20 to promote the cardiomyocyte differentiation. It is also involved in epicardial EMT which is regulated by both TGF-beta and BMPs. The SMADs negatively regulate TGF-beta [227, 228, 229, 230, 231].
Role in proliferation, differentiation and in tumorigenesis:
BMPs have dual role in tumorigenesis. It is capable of acting both as tumor suppressor and the promotor of tumor development. This is based on microenvironment and overall profile of governing key regulatory genes. Such as the absence of BMPs cause the progression of colorectal carcinoma. In Barrett's esophagus, the BMP-signaling pathway is upregulated. BMP4 also contribute to neural development. BMPs interact with K-RAS and are upregulated in NSCLC [232]. The BMPs are also involved in adult tissue homeostasis. In cardiogenesis, BMP2 causes the differentiation of CPCs. Similarly, BMP10 reduces the cardiomyocyte proliferation potential [233]. The gene expression of BMP2 in cardiac cushions causes EMT myocardial patterning. The role of BMPs is influenced by the microenvironment [234]. The BMP-signaling pathway also acts on the progenitor genes. It promotes the gene expression of Oct-4 and Nestin. They are among the key genes involved in stem cells [235]. Another fascinating feature of BMPs includes their interactions with tumor suppressors such as p53, p21, SMADs and cause repression of TGF-beta. When the p53 is mutated, this upregulates the Wnt-signaling pathway. As a result of Wnt-pathway upregulation, the interconnected loop of BMP signaling becomes dysregulated [236, 237]. BMPs have been found to act as tumor suppressors in RCC, GBM, esophageal adenocarcinoma, prostate adenocarcinoma, diffuse gastric adenocarcinoma and others [238, 239].
The role of BMPs in hepatocellular carcinoma is different and of immense significance as these cells have vast regenerative potential. Here BMPs contribute towards G1 to S transitions through cyclins [240, 241, 242].
Possible role in cardiac myxoma:
The BMPs may possibly have a very significant role in cardiac myxoma development as it is involved in cardiomyocyte differentiation in the process of cardiogenesis. As it is also involved in limiting the cardiac regenerative potential, it may have enormous influence in defining the nature of cardiac myxoma. The BMPs may have an important contributing role in CM development. Further studies should be conducted to evaluate the role of BMPs in CM development. [1,7, 19]
- Notch signaling pathway
Role in cardiogenesis and key interactions:
It is involved in cardiomyocyte proliferation, differentiation, cell fate specification and patterning. Its specific role depends on its interactions with other key regulatory genes such as it interacts with BMP2 to promote cardiomyocyte differentiation. Similarly, it interacts with activins and PI3K/AKT pathway to promote mesenchymal state in CPCs. The notch pathway interacts with p21, c-myc, snail1/2, TGF-beta and in EMT it interacts with Dll4, Jag1, BMP2, Alk3/6 and other key regulatory genes [243, 244, 245, 246]. The notch-bmp2-snail1 complex plays key role in Epicardial to Mesenchymal Transition (EMT). In EMT, it also works through important interactions with snail1/2-TGFbeta. In SHF, Notch regulates BMP4 and FGF8 gene expression [247, 248, 249].
Role in proliferation, differentiation and in tumorigenesis:
The notch pathway in cancer contributes to the stemness of cancer stem cells [250]. It interacts with proto-oncogenes and inflammatory pathways. It also has strong cross-talk with FGF and Wnt-signaling pathways [251]. Notch has key interactions with many tumor suppressors such as PTEN, P53, P21 and others. The network of these key cross-talks governs the direction of cell fate, and dysregulations in such key regulators contribute to the disease development including cancers [252, 253].
Possible role in cardiac myxoma:
The role of notch is also of enormous significance and may have possible implications in cardiac myxoma origins because of its interactions with key cardiac TFs such as Isl1 and Mef2c. The Notch expression increases the postnatal cardiac survival and in development contributes to the proliferation of CPCs. The notch signaling pathway governs cardiac tissue renewal by maintaining the CPCs in committed state. This pathway may have an important contributing role in CM development. Further studies should be conducted to evaluate the role of Notch in CM development. [1,7, 19]
Occurrence of Cardiac Myxoma in Carney Complex, pointing towards the significance of findings in this study:
In carney complex, there are mutations in a gene that acts also as a tumor suppressor PRKAR1A, located on chromosome 17. The role of carney complex is of immense significance as it also causes development of cardiac myxoma. The carney complex also causes other tumors including thyroid tumors because of increase in gene expression of RET/PTC2 signaling, multiple endocrine neoplasias and myxomas [254]. Mutations in PRKAR1A lead to the onset of dysregulated c-AMP protein kinase A signaling. The cardiac myxomas occur in 20-40% of cardiac myxoma patients and can occur in many chambers [255]. The nature of carney complex also points towards the origin of cardiac myxoma as postulated in this study. This signifies how important is the role of differentiation-related genes/tumor suppressors in the maintenance of cell type specific gene expression in cardiomyocytes. It also signifies how the defects in such key regulatory genes such as PRKAR1A can result in switching of cardiac cells towards a mesenchymal-like progenitor state present in cardiac myxoma [256, 257]. The CM development in carney complex also signifies the role of tumor suppressors and the differentiation-related genes/TFs in maintenance, homeostasis of cardiomyocytes and also in tumorigenesis.