Cancer Therapy Vol 7, 77-96, 2009

Cardiac myxoma: molecular markers, critical disease pathways, drug targets, and putative targeting miRs

Research Article

Debmalya Barh*, Sanjeeb Parida

Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, WB, India

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*Correspondence: Debmalya Barh, Ph.D., Centre for Genomics and Applied Gene Technology, IIOAB (www.iioab.webs.com), Nonakuri, Purba Medinipur, West Bengal -721172, India; Tel: +91-944-955-0032; e-mail: dr.barh@gmail.com

Key words: Biomarkers, cardiac myxoma, critical disease pathway, drug targets, key nodes, let-7, microRNA

Abbreviations: Alpha-Smooth muscle actin, (α-SMA); Calretinin, (CALB2); Canary complex, (CC); Cardiac homeobox gene, (CHG); computed tomographic, (CT); Desmin, (DES); Endothelin, (ET1); Fibromodulin, (FMOD); gradient recalled echo, (GRE); high throughput, (HTP); Lethal-7, (let-7); magnetic resonance imaging, (MRI); microRNA, (miR); Myosin head motor domain, (MYH8); Neuron-specific enolase, (NSE); Neuron-specific enolase, (NSE); Phospholipid transfer protein, (PLTP); sporadic cardiac myxoma, (CM); Thrombomodulin, (THBD); Thymidine phosphorylase, (TYMP); Vimentin, (VIM)

Received: 21 October 2008; Revised: 11 January 2009

Accepted: 27 January 2009; electronically published: February 2009

Summary

Left atrial sporadic cardiac myxoma (CM) is the commonest benign tumor of heart. Till now no drug is available and surgery is the only treatment option which frequently evokes post-surgery complaints mainly recurrence and other cardiac problems. Therefore, there is a dare need of alternative treatment options. But due to the rarity of the disease, less characterization, and unknown biology; drug targets are not yet identified. In this study, using approaches from bioinformatics, molecular markers in CM have been identified and subsequent critical disease pathways have been developed. Then analyzing pathway networks, potential drug targets are identified. Finally, various miR analysis databases and tools are used to identify potential miRs those may block the entire critical disease pathway. Analysis shows that seven groups of molecular markers and five critical disease pathways (either solitary or in interplay) are involved in CM development. 37 key nodes and several potential drug targets have been identified and combination of let-7, miR-125, miR-205, miR-214, miR-217, and miR-296 found to target maximum key molecules and predicted to have potentiality to disrupt the entire critical disease pathway network. The precise role of these miRs in CM development and their potentiality in CM therapy need to be thoroughly studied keeping in mind the challenges in RNA based therapeutics. Similarly these identified drug targets are required to be experimentally evaluated with novel targeting agents. In this article we have also provided an overview of cardiac myxoma. But because of the rarity of the disease, a systematic and multi-institutional approach is essential for better understanding of the molecular pathogenesis and subsequent drug development.

I. Introduction

A. Cardiac myxoma epidemiology

Primary cardiac tumors are very unusual (Reynan, 1996) that may constitutes less than 0.1% of all cancers (Lam et al, 1993). 75% of cardiac tumors are benign in nature and rest are malignant. 75% malignant cardiac tumors are sarcomas (Vander Salm, 2000; Laissy et al, 2004) and most frequent are Angiosarcoma (33%), Rhabdomyosarcoma (21%), Mesothelioma (16%), Fibrosarcoma (11.3%), Lymphoma (6%), Osteosarcoma (4%), Thymoma (3%), Neurogenic sarcoma (2%), Leiomyosarcoma (< 1%), Liposarcoma (< 1%), and Synovial sarcoma (< 1%) (McAllister et al, 1978; Lam et al, 1993; Vander Salm, 2000; Fernandes et al, 2001; Piazza et al, 2004). Reports suggest that malignant cardiac tumors may cause 20 times higher death rate than that of the primary tumors (Salcedo et al, 1992; Lam et al, 1993; Silvestri et al 1997).

Cardiac myxoma is the most common benign tumor of heart which accounts for 50-85% of all primary cardiac tumors (Holley et al, 1995; Reynen, 1995; Roberts, 1997; Bossert et al, 2005; Roschkov et al, 2007; Figueroa-Torres et al, 2008). 90% of the myxomas are sporadic and only around 7% are heritable (familial myxoma), referred as Carney complex (CC) that is transmitted in an X-linked autosomal dominant manner and characterized by primary pigmented nodular adrenocortical disease along with cutaneous pigmentous lentigines and hypercortisolism (Carney, 1985; Carney et al, 1986; Reynen, 1995; Carney and Ferreiro 1996). 94% of CMs are generally solitary in nature and the incidence is highest in age groups between 30 and 60 years (Carney, 1985). The Caribbean population takes mean age of 51.49 years to develop clinical symptoms (Figueroa-Torres et al, 2008). According to Yu et al, (2007) age group of 70-79 years is exclusively myxoma and age group of 0-9 years does not show CM. But recently one report suggests that right ventricular myxoma can occur at the age of 2 years that can block pulmonary artery (Kumagai et al, 2008).

Familial myxomas are more likely occur in young age irrespective of sex and 22% of them originate form atrium or ventricle and are generally multicentric (van Gelder et al, 1992; King et al, 1993; Burke and Virmani, 1993; Kennedy et al, 1995). CM is more common in women than men (Pinede et al, 2001) and 90% of left atrial myxomas in women have been found between the age group of 50 and 70 years (Shapiro, 2001).

B. Location of cardiac myxoma

Various studies have demonstrated that myxomas can occur in any compartment of the heart. Sporadic atrial myxomas can originate from anywhere within the atrium including the appendage but generally arise from the interatrial septum of the fossa ovalis border (Reynen, 1995). According to reports 60-86% CMs are left atrial, 15-28% right atrial, 8% right ventricular, 1.6-8.5% biatrial, and 1.6% multifocal (Reynen, 1995; Reynen, 1996; Pinede et al, 2001; Grebenc et al, 2002; Burke and Virmani, 1993; Butany et al, 2005; Irani et al, 2008; Rathore et al, 2008). Ventricular myxomas are mainly found in women and children. Left ventricular myxomas normally arise from the free wall and left ventricle. Mitral valve myxoma is rare (Rajani et al, 2008). Though biatrial and multicentric myxomas generally originate from atrial septum and frequent in familial cases (Imperio et al, 1980); sporadic case is also in report (Shaikh et al, 2008).

C. Symptoms and associated diseases

Symptoms of CM mainly depend on the location, size, and mobility of the neoplasm and 10 to 15% of patients are asymptomatic (Reynen, 1995; Pinede et al, 2001, Butany et al, 2005; Patanè et al, 2008). CMs may be lethal due to their strategic position (Gonzales et al, 1980).

Weight loss, loss of appetite, unusual fever, lethargy, fatigue, anorexia, painful erythema, upregulation of gammaglobulin, chronic anemia, anorexia, and facial edema can be considered as constitutional symptoms in cardiac myxoma (Glasser et al, 1971; Reynen, 1995; Tok et al, 2007; Sotokawa et al, 2008). Multiple myxomas originating from mitral leaflets blocking the left ventricular outflow have been reported by Ozcan and colleagues in 2008. Mitral valve obstruction have been noticed in 53% patients and respectively 56% and 50% cases of calcification and cardiomegaly have been found in right atrial myxoma (Grebenc et al, 2002). Right atrial myxomas generally have broad-based attachments and occasionally found calcified than that of the left atrial myxomas (St. John et al, 1980; Kennedy et al, 1995). They rarely arise from inferior vena cava (Juneja et al, 2006) and superior part of the interatrial septum (Duran and Ozkan, 2008).

In case of left atrial myxoma, most common clinical symptoms are dyspnea (Becker et al, 2008) and embolic complications. According to some reports specific symptoms are dyspnea (54-71%), cardiac auscultation (71%), increased erythrocyte sedimentation rate (51%), neurological symptoms (50%), embolism (27%), and atrial fibrillation (19%) (Acebo et al, 2003; Sultan et al, 2006). High fever (Uchino et al, 2002), and irregular mysterious fever for years (Falasca et al, 2008) are also have been reported. Embolisms have been observed in 30-50% cases of CM (Orlandi et al, 2005). Rare symptoms like chronic progressive renal failure and neuroinfection (Dewilde et al, 1983; Janion et al, 2008), rheumatic mitral stenosis (Seagle et al, 1984), severe dyspnea on exertion, tricuspid valve injury (Hirota et al, 2004), low cardiac output syndrome (Pelczar et al, 2004), myocardial infarction (Demir et al, 2005), left ventricular dysfunction (Kuźniar et al, 2007), cerebral artery infarction and embolization to eye and kidney (Yeh et al, 2006), cerebral infarction and embolism (Pradhan and Acharya, 2006; Thielke et al 2008), sudden death due to acute pulmonary embolism (Sato et al 2008), ischemic stroke (Yoo and Graybeal, 2008), Sepsis (Janion et al, 2008), and ventricular fibrillation (Attar et al, 2008). Myxomas infected with Streptococcus viridans (Dawson et al, 1988; Karachalios et al, 2004), S. oralis (Uchino et al, 2002; García-Quintana et al, 2005; Janion et al, 2008), and Enterococcus faecalis (Leone et al, 2008) complicates the disease. Caribbean myxoma population have been reported to show congestive heart failure (35%), chest pain (18%), and neurologic symptoms (14%) (Figueroa-Torres et al, 2008). Neurological symptoms are aphasia, hemiparesis, eye sight problem, and progressive dementia. Dyspnoea, renal emboli, pulmonary embolism (Mattle et al, 1995) and neovascularization (Fueredi et al, 1989; Van Cleemput et al, 1993) are also in report. Severe complications includes pulmonary hypertension due blockage of the right atrioventricular ostium or to embolism or Budd-Chiari syndrome with severe abdominal pain (Tok et al, 2007). Malignancy, metastasis, and recurrence have been found in many cases (Gerbode et al, 1967; Hannah et al, 1982; Gray and Williams, 1985; Markel et al, 1986; Shinfeld et al, 1998; Hou et al, 2001; Kurian et al, 2006). Atrial myxoma frequently results in heart failure, stroke, and rheumatologic symptoms (Carney, 1985; McCarthy et al, 1986; Molina et al, 1990; Gawdzínski et al, 1996; Centofanti et al, 1999; Kapusta et al, 2007; Yanagawa et al, 2008, Detko-Barczyńska et al 2008), dyspnea, chest pain, fever, syncope, tricuspid regurgitation, ventricular failure, systemic embolism, and positional syncope (Chu et al, 2005; Bakkali et al 2008). While large left atrial myxomas correlate with constitutional symptoms, congestive heart failure, syncope, and mitral valve disease; smaller myxomas are generally associated with embolization (Patanè et al, 2008). Several asymptomatic cases are also recently reported those may be associated with neovascularization and ossification (Detko-Barczyńska et al, 2008; Panagiotou et al, 2008).

20% of familial myxomas are complex myxomas (McCarthy et al, 1986) those generally show adrenocortical nodule hyperplasia, Sertoli cell and pituitary tumors, multiple myxoid breastfibroadenoma, cutaneous myomas, and facial or labial pigmented spots (Carney, 1985; Carney et al, 1986).

CMs are reported to associate with Graves' disease (Suzuki et al, 1999), arthrogryposis (Veugelers et al, 2004), myocardial infarction (Namazee et al, 2008; Patanè et al, 2008), neuroblastoma therapy (Hill et al, 2008), Gerstmann's syndrome (Sakellaridis et al, 2008), and hemangioblastoma (Yanagawa et al, 2008).

D. Histology

Though the myxoma is histologically benign (Reynen, 1995), myxoma population is found to be heterogeneous in nature. CMs are generally grossly, gelatinous and villous in appearance, rounded or oval shaped with a smooth or lobular surface, 5 -15 cm in diameter, covered with thrombus, and rapidly growing. Most CMs are stalked and sessile forms are uncommon. Papillary myxomas are fragile and gelatinous those occasionally form emboli (Reynen, 1995; Pinede et al, 2001; Yeh et al, 2006, Becker et al, 2008). Left atrial myxomas have reported to show an average 1.8 or 5.7 cm/year growth rate (Malekzadeh and Roberts, 1989; Lane et al, 1994). In general, histology varies depending on site, clinical appearance, age and sex of the patient. Although according to Merkow and colleagues in 1969 mitosis is unlikely in myxoma, nuclear mitosis in myxoma are also in report (Burke and Virmani, 1993; Kusumi et al, 2008). Surface thrombus (41%), fibrosis (41%), mitotic activity (23%), calcification (20%), gamma bodies (17%), ossification (8%), extramedullary hematopoiesis (7%), mucin-forming glands (3%), atypical cells those simulates malignancy (3%), and thymic rests (1%) have been reported in corresponding percent cases. Calcification and fibrosis are more likely found in respectively right atrial and nonembolic myxomas. Embolic tumors are often thrombosed and widely myxoid in nature with an irregular frond-like surface (Burke and Virmani, 1993). Electron microscopy shows various types of mesenchymal multipotential cells in different differentiation stages and electron dense granules and stray collagen fibers localizes in the matrix (Chopra and Sharma, 1983). Myxomas are generally polypoid and show polygonal cells having oval nuclei and eosinophilic cytoplasm, capillary channels, myxomatous tissue, and coagulation necrotic bodies embedded in an acid-mucopolysaccaride myxoid matrix (Reynen, 1995; Rahmanian et al, 2007; Kusumi et al, 2008). The matrix also found to contain smooth muscle cells, reticulocytes, collagen, elastin fibers, and blood cells in various proportions. The stalk can be a solid mass consisting of spindle-shaped tumor cells with hypercellular proliferation activities (Kusumi et al, 2008). Cysts, extramedullary hematopoesis focai, and hemorrhage lesions are reported to scattered throughout the matrix (Prichard, 1951; Kaminsky et al, 1989; Carney et al, 1986). Calcium and metastatic bone deposits and glandular-like structures have been reported in 10% cases (Kaminsky et al, 1989; Carney et al, 1986).

E. Diagnosis

Echocardiography is frequently used and most reliable diagnostic method for early detection (Reynen, 1995; Becker et al, 2008; Yoo and Graybeal, 2008) and for familial variant of cardiac artrial myxomas (Sun and Wang, 2008). But differential diagnostic approaches are required for CM diagnosis those must include valvular heart disease, disturbances cardiac rhythm, cardiac insufficiency, cardiomegaly, syncope, systemic or pulmonary embolism, and bacterial endocarditis. Transesophageal (Reynen, 1995) along with transthoracic echocardiography is most effective and is recommended first to know the size, localization and mobility of the tumor mass (Rahmanian et al, 2007). As the atrial thrombi often mimic the echocardiographic features of atrial myxoma and also echocardiography cannot distinguish myxoma and other cardiac mass (Perez de Isla et al, 2002), additional approaches along with echocardiography such as thoracic computed tomographic (CT) scan (Mattle et al, 1995), coronary angiography for tumors localizes to right coronary atrial branches and neovascularization (Van et al, 1993; Janas et al, 2006; Roth et al, 2006; Rahmanian et al, 2007) is required to be performed. Heterogeneous or hypoattenuated spherical or ovoid lesions with lobular borders and point of attachment can be observed in CT scan and magnetic resonance imaging (MRI). Multiplanar and Cine gradient recalled echo (GRE) MR imaging, can provide precise location, size, and point of attachment of myxoma lesions that can assist in surgery (Grebenc et al, 2002). MRI can be used to precisely characterize myxoma and cardiac thrombi. For myxoma MRI specifically shows the typical histological signs like hypointensity of the mass, high extra cellular water content, heterogeneity, necrotic lesions, calcification, and hemorrhage. Gadolinium-enhanced MRI can be used to find mass-perfusion (Rahmanian et al, 2007). Recently X-ray (Sato et al, 2008) and ultrasound (West and Kaluza, 2008) have reported for diagnosis.

F. Treatments and complications

Till now surgery is the only available treatment option and alone chemotherapy is not satisfactory. Surgical excision of the myxoma is not only costly and risky it also shows several post-surgery complaints. Although in certain cases surgery prevents recurrence (Durgut et al, 2002), lowers mortality rate, and gives good long-term outcome (D'Alfonso et al, 2008), post surgery problems like transient ischemic attacks and a stroke with persistent neurological deficit, complete atrioventricular block, and myocardial infarction (Scrofani et al, 2002), respiratory failure (Bakaeen et al, 2003), stroke (Lad et al, 2006), cardiac tamponade (Swartz et al, 2006), and recurrence (Vohra et al, 2002; Hermans et al, 2003; Macarie et al, 2004; Kojima et al, 2005; Ito et al, 2006) have been reported in many cases. Incidence of recurrence has been reported to be 1-3% in sporadic, 12% in familial, and 22% in complex form of myxomas (Etxebeste et al, 1998). Second recurrence is unlikely except few reports (Duveau et al, 1994; Roldan et al, 2000). Recurrence from left atrium and right ventricle to respectively left ventricle and left atrium is recently reported (Rathore et al, 2008). Recurrence occurs due to incomplete removal of the original tumour, familial predisposition, intracardiac embolic fragment implantation, and existence of pretumoural foci in the myocardium (Hermans et al, 2003). Endoscopically assisted surgical excision with supplementary cryoablation can be effective in recurrent cases (Rathore et al, 2008). Biatrial myxoma can be removed by surgery using a cardiopulmonary bypass through a midline sternotomy (Irani et al, 2008). Conventional transeptal, left and right atriotomy, and combined transeptal and atriotomy can be performed for effective and long term survival (Becker et al, 2008).

G. Objective

To avoid costly and risky surgery, post-surgery complications, and moreover, to develop alternative treatment; the biology and drug targets of cardiac myxoma should be thoroughly studied. In this research, based on available data; molecular biomarkers, critical disease pathways, and drug targets of CM have been identified. Next, an effort has been made to target those critical components of the constructed pathway with minimum numbers of naturally occurring miRs to block the entire network, which might be a potential therapeutic strategy in CM.

II. Materials and Methods

A broad bioinformatics approach was taken into consideration. Pubmed, Elsevier, and Medline literature databases were screened for articles describing sporadic cardiac mixoma related genes, proteins, markers etc. Data obtained from searches were classified into four groups viz. expressed, down-regulated, up-regulated, and highly up-regulated in malignant cases. Frequently expressed and up-regulated genes are considered for critical disease pathway construction. Initially, Osprey 1.0.1 powered with human GRID database (http://biodata.mshri.on.ca/osprey/servlet/Index) is used to find out protein-protein interaction and then a general myxoma disease pathway is constructed by mining various pathways from Invitrogen, KEGG, BioCarta, Ambion, and Cell Signaling pathway databases using identified genes from literature mining (pathway not shown). Signaling network, key nodes, and up and down-stream target analysis are done following methods of Barh and Das in 2008. Considering all results, the final critical disease pathway has been drawn using CellDesigner4.0beta (http://systems-biology.org/software/celldesigner). Identification of minimum number of miRs to block the entire critical disease pathway is based on as described by Barh and colleagues in 2008.

III. Results

A. Molecular markers in cardiac myxoma

Literature screening shows that, mutations in PRKAR1A gene are mostly found in familial cardiac myxomas (Carney complex) but not in sporadic cases (Casey et al, 2000; Kirschner et al, 2000; Kirschner et al, 2000; Fogt et al, 2002; Aspres et al, 2003; Kojima et al, 2005; Wilkes et al, 2005; Puntila et al, 2006). Therefore, detection of PRKAR1A mutation can help in disease prognosis (Imai et al, 2005). Microsatellite instability on chromosome 17q (Sourvinos et al, 1999) and missense mutation in the Myosin head motor domain (MYH8) (Veugelers et al, 2004) have been found in few cases of CC.

Sporadic cardiac myxoma rarely shows mutation. P53 mutation is not associated with CM and only one case of Kras mutation (Gly 12 Asp) has been reported by Karga and colleagues in 2000. Cardiac myxomas do not express Myoglobin (Johansson, 1989), P53 and Bcl2 (Suvarna SK, and Royds, 1996), Stem cell factor, Granulocyte colony-stimulating factor, Hepatocyte growth factor, and ET3 (Sakamoto et al, 2004), and ErbB3 and WT1 (Orlandi et al, 2006).

CMs show expression of wide range of molecular markers of various functionalities. CMs express F8, Desmin (DES), Vimentin (VIM), Cytokeratin (CAM 5.2 and AE1/AE3), and S100 (S100A1) (Johansson, 1989), Lu-5, CAM 5.2, VIM, and Neuron-specific enolase (NSE) (Curschellas et al, 1991), Factor XIIIa (Berrutti et al, 1996), CD34 and CD31 (Farrell et al, 1996), PCNA, MIB1, nm23, and RB1 (Suvarna et al, 1996), CEA, and CA19.9 (Lindner et al, 1999), Protein 9.5, S100, and NSE (Pucci et al, 2000), Ras and P21 (Karga et al, 2000), Calretinin (CALB2) (Terracciano et al, 2000), Thrombomodulin (THBD) and CALB2 (Acebo et al, 2001), VIM, S100, and NSE (Oyama et al, 2001), Nkx2.5/Csx, GATA4, Cardiac homeobox gene (CHG), and eHAND (Kodama et al, 2002), Chemotactic protein-1 (CCL2), Thymidine phosphorylase (TYMP), and CC chemokine receptor-2 (CCR2) (Zhang et al, 2003), ANXA3, Phospholipid transfer protein (PLTP), Tissue inhibitor of metalloproteinase 1 (TIMP1), Secretory leucocyte protease inhibitor (SLPI), SPP1, Fibromodulin (FMOD), SOX9, and CALB2 (Skamrov et al, 2004), MUC2 and MUC5AC (Chu et al, 2005), and Alpha-Smooth muscle actin (α-SMA), CD34, Sox9, Notch1, NFATc1, Smad6, MMP1 and MMP2 (Orlandi et al, 2006).

While downregulation is observed for vascular myosin heavy chain isoform (SM2) (Suzuki et al, 2000), in several cases upregulation has been observed for nonmuscle-type vascular myosin heavy chain isoform (SMemb) (Suzuki et al, 2000), CXC chemokines, IL8, Growth-related oncogene-α, Endothelin (ET1) and its precursor Big ET1 (Sakamoto et al, 2004), MIA and PLA2G2A (10 fold higher) (Skamrov et al, 2004), MT1-MMP, Pro-MMP2, TIMP2, and Pro-MMP-9 in embolic myxomas (Augusto et al, 2005), MUC1 (Chu et al, 2005), and C-reactive protein and Gammaglobulin (Endo et al, 2006). Highly proliferative, angiogenic, and malignant myxomas exhibit over-expression of IL6 (Hirano et al, 1987; Seino et al, 1993; Parissis et al, 1996; Mendoza et al, 2001), VEGF, PCNA, FGFβ, FGFR1, VEGF, VEGFR1 (flt-1), and VEGFR2 (Kono et al, 2000; Fujisawa et al, 2002; Sakamoto et al, 2004). Table 1 represents a list of frequently up-regulated markers in cardiac myxoma.

B. Protein-protein interactions and functional groups of molecular markers

The Osprey interaction map (Figure 1) shows that several groups of proteins consisting of complicated mitogenic and cardiac developmental pathways are involved in CM pathogenesis. Key proteins those are expressed in myxoma are found to be involved in G-protein signaling (CCL2, CCR2, EDN1, IL8), heart development (EDN1, GATA4, HAND1, MIB1, NFATC1, NKX2.5, NOTCH1, SOX9), angiogenesis (FGF2, FGFR1, VEGFR2, NOTCH1,THBD, VEGFA), cell proliferation (FGF2, FGFR1, IL6, IL8, VEGFR1, VEGFR2, MIA, NME1, NOTCH1, SOX9, TIMP1, TIMP2, VEGFA), cell adhesion (F8, MIA, THBD), cell cycle (PCNA, RB1), and cell migration and metastasis (VEGFR2, MMP2, MMP9, THBD, VEGFA, VIM). Osprey interaction map also shows that these proteins interact with various proteins those are also involved in processes like heart development and tumorigenesis (Table 2). Specific groups (same for biological function) of proteins are found to interact with other groups of proteins by connecting molecules. For example, groups of heart developmental proteins where key proteins are GATA4, HIND1, and SOX9 link to growth receptor signaling pathway through KPNB1. Similarly NOTCH1 signaling pathway of heart development is connected to growth receptor signaling pathway through MUC1 (Figure 1). Reported individual case specific markers like F8, THBD, FMOD, PLA2G2A, CCL2, and CCR2 are connected to the entire network through MMP2 (Figure 1).

Table 1. Frequently upregulated proteins in sporadic cardiac myxoma.

Molecular markers	% of Cases	References
Protein gene product 9.5	94%	Angela et al, 2000
S100A1	88%	Angela et al, 2000
Neuron-specific enolase	56%	Angela et al, 2000
CALB2	100%	Terracciano et al, 2000
THBD	82.6%	Acebo et al, 2001
Calretinin	73.9%	Acebo et al, 2001
bFGF	73.3%	Fujisawa et al, 2002
FGFR1	67.7%	Fujisawa et al, 2002
Sox9	100%	Orlandi et al, 2006
Notch1	87.5%	Orlandi et al, 2006
NFATc1	37.5%	Orlandi et al, 2006

Table 2. Osprey protein-protein interaction. Only key proteins and their interacting partners and functions are listed. NA denotes for data not available. Proteins in bold letters are directly involved in cardiogenesis or cardiac function or cardiac myxoma. Annotations are taken form Human GRID database associated with Osprey tool.

Protein	Functions	Interactions	Comments
ACTA2 (Alpha-cardiac actin)	ATP binding, muscle development.	VDBG+CCTs+TMSB4X+CFL1+DNASE1+MYL1	VDBG: vitamin D binding; CCT: cell cycle regulation; TMSB4X: cellular component organization and biogenesis; CFL1: Rho signaling, anti-apoptosis; DNASE1: apoptosis; MYL1: muscle development
ANXA3 (Annexin III)	Diphosphoinositol-polyphosphate diphosphatase and phospholipase A2 inhibitor.	REG3A	REG3A: multicellular organismal development, heterophilic cell adhesion, cell proliferation.
CALB2 (Calbindin 2)	NA	TUBA1+KRT1	TUBA1: bone development; KRT1: epidermis development.
CCL2 (Monocyte chemoattractant protein-1)	G-protein signaling, apoptosis.	FY+CSPG2+CCR5+ CCR2	FY: GPCR pathway; CSPG2: cell adhesion, multicellular organismal development; CCR5: GPCR pathway; CCR2: GPCR pathway.
CCR2 (MCP-1 receptor)	GPCR pathway.	CSPG2+CCR5+ CCL2+CCL7+JAK2+IFR2	CCL7: chemokine activity; JAK2: cell cycle regulation, myeloid cell differentiation, mesoderm development, apoptosis, IFR2: NA
CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5)	NA	HNRPM+EWSR1	HNRPM: RNA splicing; EWSR1: regulation of transcription.
CRP (C-Reactive Protein)	C-Reactive Protein	NA	NA
DES (Desmin)	Muscle development and contraction, heart contraction.	DSP+S100B+S100A1+SYNC1	DSP: epithelial to mesenchymal transition, epidermis development; S100B: cell proliferation; S100A1: regulation of heart contraction, cell communication; SYNC1: NA
EDN1/ET1 (Endothelin 1)	GPCR Pathway, heart development.	NPPC+EDNRA+ADM	NPPC: vasoconstriction; EDNRA: heart development, embryonic development; ADM: heart development, induction of cell proliferation.
F8 (Coagulation factor VIII)	Cell adhesion.	F9+CANX+VWF+F10+PROC	F9: blood coagulation; CANX: angiogenesis; VWF: platelet activation, cell adhesion; PROC: negative regulation of apoptosis, chymotrypsin activity.
FGF 2 (Fibroblast growth factor 2)	Ras protein signaling, MAPKK activation, cell proliferation, angiogenesis.	FGFR1	FGFR1: cell proliferation, morphogenesis.
FGFR1 (Fibroblast growth factor receptor 1)	Negative regulation of apoptosis; cell proliferation, angiogenesis, MAPKKK cascade.	FGF5+SHC1+KPNB1+SHB+FGF	FGF5: FGF signaling, cell cycle regulation, cell proliferation; SHC1: EGFR signaling, MAPK activation, induction of cell proliferation; FGF2: (See previous)
FMOD (Fibromodulin)	Transforming growth factor.	TGFB1+TGFB2+ TGFB3	TGFB1: anti-apoptosis, regulation of cell cycle and proliferation, epithelial to mesenchymal transition, exit from mitosis, NF-kappaB activation; TGFB2: cytokine activity, induction of cardioblast differentiation, cardiomyocyte proliferation and cardioblast differentiation, heart development, heart contraction, mesoderm formation, angiogenesis, TGFB production, anti-apoptosis; TGFB3: activation of MAPK activity, epithelial to mesenchymal transition, cell cycle regulation.
GATA4 (GATA binding protein 4)	Transcription factor, gastrulation, embryonic heart tube pattern formation.	NR5A1+ZFPM2+MAPK3+MEF2C+NFATC4 (NFAT3)+FOS+TBX5+NKX2-5+HAND2	MAPK3: cell cycle regulation, organ morphogenesis; MEF2C: heart development, blood vessel development and remodeling; NFATC4: transcription factor, heart development, FOS: transcription factor, cell proliferation; TBX5: transcription factor, pericardium development, cardioblast differentiation, heart development, inhibition of cardiac muscle cell proliferation; NKX2-5: transcription factor, cardiac muscle development and differentiation, adult heart development, heart looping, embryonic heart tube development; HAND2: transcription factor, adult heart development, heart looping, angiogenesis.
HAND1 (Basic helix-loop-helix transcription factor HAND1)	Heart development, cell differentiation, mesoderm formation, multicellular organismal development.	HAND2+MYOD1+PRKACA+PRKCA+TCF3+PPP2R5D	HAND2: (See previous); MYOD1: myoblast differentiation and fate determination; PRKCA: regulation of heart contraction and cell cycle, induction of apoptosis.
IL6 (Interleukin-6)	Cytokine, negative regulation of apoptosis, regulation of cell proliferation.	PTHLH+IL6R	PTHLH: G-protein signaling, epithelial cell differentiation, positive regulation of cell proliferation; IL6R: cell proliferation.
IL8 (Interleukin-8)	Chemokine , GPCR signaling, regulation of cell proliferation, angiogenesis, regulation of cell adhesion.	FY+GNA12+SDC1+IL8RA+CCL4+IL8RB	FY: chemokine, GPCR pathway; IL8RA: GPCR pathway; CCL4: chemokine, cell adhesion, cell polarity; IL8RB: chemokine, GPCR pathwa, positive regulation of cell proliferation.
VEGFR1	Angiogenesis, cell proliferation.	FGF5+SHB+SHC1+FGF+GRB2	FGF5: FGF signaling, cell proliferation; SHB: angiogenesis; SHC1: EGFR signaling, MAPK activation, positive regulation of cell proliferation; GRB2: Ras and EGFR signaling.
KDR/VEGFR2 (Kinase insert domain receptor)	Endothelial cell differentiation, hemopoiesis, angiogenesis, cell migration.	VEGFA+FLT1/ VEGFR1+ SHB +SHC1+GRB2	(See previous and later)
MIA (Melanoma inhibitory activity)	Cell proliferation, cell-matrix adhesion.	FN1	FN1: cell migration, cell adhesion.
MIB1 (Mind bomb homolog 1)	Heart looping and development, blood vessel development.	DAPK1+DAB2	DAPK1: anti-apoptosis; DAB2: cell proliferation.
MMP2 (Matrix metalloproteinase 2)	Collagen catabolism, blood vessel maturation.	CCL7+TGFB1+THBS1+TIMP2+COL18A1+ITGAV+MMP17+CXCL12+MMP14+PSMA7+MMP8	THBS1: negative regulation of angiogenesis, cell adhesion; TIMP2: negative regulation of cell proliferation; COL18A1: organ morphogenesis, negative regulation of cell proliferation, cell adhesion; ITGAV: cell-matrix adhesion, blood vessel development, integrin-mediated signaling pathway; CXCL12: cell adhesion, GPCR pathway; MMP14: endothelial cell proliferation, focal adhesion formation, angiogenesis, tissue remodeling, cell migration.
MMP9 (Matrix metalloproteinase 9)	Skeletal development, apoptosis, extracellular matrix organization and biogenesis, collagen catabolism.	THBS1+LCN2+TIMP3+THBS2+COL1A1+FN1+MMP7+CD44+RECK+COL4AS	THBS1: (See previous); TIMP3: transmembrane receptor protein tyrosine kinase signaling pathway, induction of apoptosis by extracellular signals; THBS2: cell adhesion; COL1A1: epidermis development; FN1: (See previous); CD44: cell-matrix adhesion; RECK: extracellular matrix organization and biogenesis, negative regulation of cell cycle.
MUC1 (Mucin 1)	Actin binding, hormone activity.	GSK3B+ERBB2,3,4+PRKCD+APC+SOS1+CTNNB1+CTNND1+EGFR+GALNT1+VEGFC+GRB2	GSK3B: NF-kappaB binding, negative regulation of apoptosis, Wnt receptor signaling; ERBB2,3,4: positive regulation of epithelial cell proliferation, angiogenesis, heart development; PRKCD: negative regulation of cell cycle, Wnt receptor signaling, pattern formation; CTNNB1: cell adhesion, ectoderm development, negative regulation of cell differentiation, gastrulation, heart development; CTNND1: cell adhesion; EGFR: positive regulation of cell proliferation and migration; GRB2: epidermal growth factor receptor signaling pathway.
MYH8 (Myosin heavy polypeptide 8)	Muscle development.	NA
NFATC1 (NFAT transcription complex cytosolic component)	Heart development, G1/S transition of mitotic cell cycle.	PIM1	PIM1: multicellular organismal development, negative regulation of apoptosis, cell proliferation.
NKX2-5 (NK2 transcription factor related, locus 5)	Cardiac muscle development and differentiation, heart looping, embryonic heart tube development.	HIPK1+HIPK2+SRF+TBX5+GATA4	TBX5: (See previous); GATA4: (See previous); HIPK2: induction of apoptosis, positive regulation of TGF beta receptor signaling pathway; SRF: heart development and heart looping, muscle maintenance.
NME1 (Non-metastatic cells 1)	Positive regulation of epithelial cell proliferation, regulation of apoptosis.	APEX1+SET+RRAD+POLR1C+PCNA	RRAD: small GTPase mediated signal transduction; PCNA: (See later)
NOTCH1 (Neurogenic locus notch homolog protein 1)	Heart development, endoderm development, Notch signaling pathway, cell proliferation, angiogenesis, regulation of apoptosis.	GSK3B+LEF1+RBPSUH+DLL4+RELA+PSEN1+NFKB1	GSK3B (see later); LEF1: mesoderm formation, Wnt receptor signaling pathway, somitogenesis; RBPSUH: Notch signaling pathway, epithelial to mesenchymal transition, angiogenesis, heart development, hemopoiesis, cell proliferation; DLL4: angiogenesis, multicellular organismal development, Notch signaling pathway; RELA: anti-apoptosis, chemokine mediated signaling, NF-kappaB activation; PSEN1: anti-apoptosis, cell adhesion, heart development, Notch receptor processing; NFKB1: anti-apoptosis.
PCNA (Proliferating cell nuclear antigen)	DNA replication, regulation of progression through cell cycle, cell proliferation.	CCND1+HDAC1+DNMT1+CDK2+APEX	CCND1: re-entry into mitotic cell cycle, positive regulation of cyclin-dependent protein kinase activity; HDAC1: anti-apoptosis; CDK2: G2/M transition of mitotic cell cycle, positive regulation of cell proliferation.
PLA2G2A (Phospholipase A2, group IIA)	Phospholipids metabolism.	CSPG2+DCN+PLA2G1B	CSPG2: cell adhesion, multicellular organismal development; DCN: organ morphogenesis; PLA2G1B: phospholipid metabolism
RB1 (Retinoblastoma 1)	Erythrocyte differentiation, regulation of cell cycle and proliferation, androgen receptor signaling.	MYOD1+HDAC+CCND1+CDK4+E2F+MAPK9+JUN+MYC	(See previous)
S100A1 (S100 calcium-binding protein A1)	Regulation of heart contraction, intracellular signaling, nervous system development.	S100B+NIF3L1+GFAP+DES+PLN+ATP2A2+RYR1+GJA1+S100A4	S100B: induction of apoptosis, cytokine biosynthesis, cell proliferation; DES: regulation of heart contraction, muscle development, PLN: inhibition of heart contraction, cardiac muscle development, calcium ion homeostasis. ATP2A2: cell adhesion, regulation of the force of heart contraction, calcium ion homeostasis; RYR1: Apoptosis, cell motility, cell cycle regulation, muscle contraction, calcium ion transport; GJA1: NF-kappaB cascade, apoptosis, blood vessel morphogenesis, embryonic and adult heart tube development, heart looping; S100A4: epithelial to mesenchymal transition.
SMAD6 (SMAD family member 6)	Transcription factor, TGF-beta signaling.	CTBP1+SMAD1+SMURF1+MAP3K7+MAP3K7IP1	SMAD1: BMP signaling, brain development, embryonic pattern specification; CTBP1: negative regulation of cell proliferation; SMURF1: inhibition of BMP signaling, ectoderm development, cell differentiation, protein ubiquitination; MAP3K7: JNK, TGF-beta, TCR, and NF-kappaB cascades, inhibition of apoptosis, angiogenesis; MAP3K7IP1: TGF-beta signaling, embryonic development, heart morphogenesis.
SOX9 (Sex-determining region Y-box 9)	Transcription factor activity, heart development, epithelial to mesenchymal transition, regulation of cell proliferation and apoptosis.	NR5A1+MED12+MAF+TRAF2+KPNB1.	NR5A1: transcription coactivator, steroid biosynthesis; cell differentiation; TRAF2: Apoptosis, cytokine production; KPNB1: protein import into nucleus.
THBD (Thrombomodulin)	Blood coagulation, embryonic development, inhibition of angiogenesis, cell motility, cell adhesion.	PROC+ F2+CPB2+PF4+F8	PROC: (See previous); F2: blood coagulation, cell cycle regulation, apoptosis; PF4: chemokine mediated signaling; angiogenesis inhibition; F8: (See previous).
TIMP1 (Tissue inhibitor of metalloproteinase 1)	Metalloendopeptidase inhibitor, development, positive regulation of cell proliferation.	MMP3+MMP1	MMP3: collagen catabolism; MMP1: collagen catabolism.
TIMP2 (Tissue inhibitor of metalloproteinase 2)	Metalloendopeptidase inhibitor, negative regulation of cell proliferation.	MMP2+MMP14+PSMA7+MMP8	MMP2: (See previous); MMP14: (See previous); PSMA7: threonine endopeptidase, ubiquitin-dependent protein catabolism; MMP8: collagenase, proteolysis.
VEGFA (Vascular endothelial growth factor A)	Mesoderm development, anti-apoptosis, positive regulation of cell proliferation, angiogenesis, cell migration.	FGF5+FLT1+VEGFR2+NRP1+NRP2	FGF5: (See previous); FLT1: angiogenesis, cell migration, VEGFR signaling, cell differentiation. VEGFR2: (See previous); NRP1: cell adhesion, organ morphogenesis, angiogenesis, cell differentiation and proliferation, heart development, NRP2: (same as NRP1).
VIM (Vimentin)	Intermediate filament-based process, cell motility, oxygen transport.	CDH5+GFAP+DSP+NIF3L1	CDH5: cell proliferation inhibition, cell adhesion, blood vessel maturation; GFAP: intermediate filament-based process; DSP: keratinocyte differentiation; NIF3L1: unknown.

Figure 1. Osprey protein-protein interactions. Various groups of proteins showing involvement of multiple pathways in cardiac myxoma.

C. Common genes involved in heart and myxoma development

Observing the presence of heart developmental pathway in cardiac myxoma, as evident form the protein-protein interaction map, an attempt has been taken to find out the entire gene sets involved in various stages of cardiac development. Using review literatures (Srivastava and Olson, 2000; McFadden and Olson, 2002; Zaffran and Frasch 2002; Brand, 2003) and Cardiovascular Database (www.cardio.bjmu.edu.cn) cardiogenesis gene are identified and a mammalian heart development pathway has been constructed (Figure 2) with an aim to identify differentially expressed genes those are involved only in myxoma development. Unable to identify only CM related gene sub-sets form the whole pool of heart developmental genes, focus has been shifted to identify common gene set which is involved in both the processes. Result shows that, Nkx2.5/Csx, GATA4, HOX, HAND, MYOD, SOX4-6, S100, and TGF-β are involved in both heart and cardiac myxoma development. But these genes are found active at vary early stage of cardiac development mainly during transition form mesoderm. Therefore, upregulation of these genes during embryonic development may contribute to myxoma growth but as the myxoma is mostly found in the age group of 70-79 years and no cases have been diagnosed within the age group 0-9 years (Yu et al, 2007); the heart developmental pathway may not be involved in the development of CM. Therefore, a possible explanation may be an epigenetic regulation of these heart developmental genes at later stage of age that contribute to CM development. But as there is no age and stage specific high through put (HTP) expression data available for cardiac myxoma, the precise molecular mechanism of these genes or the involvement of the heart developmental pathway in CM is unclear from this analysis.

D. Cardiac myxoma critical disease pathways and drug targets

As shown in Figure 3, several pathways are found to interplay either solitary or in combination cross talking with one another in developing CM. Main critical disease pathways include CCR2, FMOD-TGFB, S100A1-FGFR, NKX2.5-GATA4-SOX9-FGFR, HAND1-GATA4, and MUC1 regulated NOTCH signaling and several mitogenic pathways. At a certain point, all pathways found to follow the growth receptor signaling pathways and MYC, FOS, and MMP9 are identified as common downstream targets. Based on key-nodes analysis, it has been found that, CCR2, TGF-β, MUC1, FGFR, EGFR, GATA4, and HAND1 are critical for the entire network. All these key nodes and their upstream regulators and downstream targets (Tables 2-3, Figure 3) those are of various molecular and biological functions are also found as potential drug targets.

Figure 2. Sequential events and gene (s) involvement in mammalian heart development.

Figure 3. Cardiac myxoma critical disease pathways. The network includes all prevalent CM markers and their normal role in the cascade. But alteration of any one of these genes will lead to CM.

Table 3. Key nodes and their upstream regulators and downstream targets.

Key nodes	Up stream regulators	Down stream targets
β-catenin	AKT, TNF	CCND1, FOS, MYC
CDK4	MYC	BRCA1, CCND1
E2F4	BRCA1	CDK1, CDK2, Cyclin A, PCNA, RB
FOS	AP1/JUN, EGF, ESR1, IFNG, IL22, SRC, STAT	ET1, FLG, FRA1, IL2, IL6, IL8, MMP1, P53
EGFR	EGF, MUC1	JUN, MAPK9, RARA
ERBB2/ HER2/neu	AR, EGF, GABPA, MUC1, MYC, SPI1	CCND1, ESR1, KRAS, MMP2, MMP9, MYC, PTEN
GATA4	HAND1, NKX2.5	FGFR, FOS, SOX9
JUN	CNK, EGF, IKK, TGF, SENP1	ESR1, MSH2
K-RAS	CTF, HER2, P53, SP1	CCND1, MMP2, MMP9
MMP2	AP-2a, ESR1, HER2, KRAS, P53	MMP9, TSP1
MUC1		β-catenin, EGFR, GRB2, NOTCH
MYC	Ap1, ap2, EGF, MAK, SP1, stat, TCF4	CDC25A, CDK4, Eif-4E, ERBB2, TERT
MMP9	HER2, MMP2, SDF1, TGF	NF-kB, TSP1, TSP2,
NF-kB	MMP9, PTEN	FOS, MYC, PCNA
PCNA	P53, IKK-a, NFkB, E2F4, P53, ESR1, AP1/JUN	DNA Ligase-1, GTBP, MSH3, POL-D and- E, RARA, RFCs
VEGF	AP1, BRCA1, ERK, SMAD, SP1, TGF	VEGF-A, VEGF-165, VEGF-145
TGF-β	FMOD	MMP2

E. microRNA: prospective therapeutics?

miRs are natural endogenous non-coding pool of small RNA molecules of 20-24 nucleotides in length which are found deregulated in several cancers and restoration of these key deregulated miRs have now showing promising results in cancer therapy. Speculating that, miRs are universally target-specific regardless the type of cancer, using similar bioinformatics approaches as described by Barh and colleagues in 2008, it has been predicted that a combination of let-7, miR-125, miR-205, miR-214, miR-217, and miR-296 can cover maximum key molecules and potentially disrupt the entire critical disease pathway of CM by targeting maximum key nodes and their upstream and downstream molecules (Table 4). But until now no high throughput (HTP) gene expression data or microRNA profile available for CM. Therefore, the precise roles of these identified putative therapeutic miRs in CM pathogenesis are yet to be experimentally identified.

Similarly, there are several challenges in RNA based therapeutics especially when microRNAs are used. Strategies like restoration of normal expression of a particular miR or inhibition of translation of a given mRNA by miR both have similar hurdles. For example, let-7 is deregulated in lung and hepatic cancers and ectopic expression or restoration of normal expression level of let-7 in these tumors have been found to represses cancer growth by inhibiting RAS, MYC, and by directly or indirectly targeting several genes of multiple cell proliferation pathways (Yanaihara et al, 2006; Inamura et al, 2007; Johnson et al, 2007; Esquela-Kerscher et al, 2008, Kumar et al, 2008; Yu et al, 2008) Similarly, induced overexpression of let-7 inhibits proliferation and growth of Burkitt lymphoma by targeting MYC and its target genes (Sampson et al, 2007), uterine leiomyoma by repressing HMGA2 (Peng et al, 2008), breast cancer by targeting HRAS and HMGA2 (Sempere et al, 2007; Yu et al, 2007), melanoma by downregulating cyclins CDK4, cyclins-A, -D1, and -D3 (Schultz et al, 2008). According to Grimm et al, (2006), ectopic AAV mediated recombinant pre-miRNAs saturated and overwhelmed XPO5 leading to the inhibition of normal cellular pre-miRNAs processing resulting liver cytoxicity and death in mouse. These facts show that a given miR can target multiple target genes. Therefore, all possible targets, immunogenic responses, and other side effects of a miR selected for therapy is required to be carefully studied. Along with the multi-gene targeting capability of a single miR, there are several other drawbacks in miR therapy as described by Barh in 2008. In brief, the foremost difficulty is the tissue specific targeted delivery. siRNA delivary methods are generally followed in delivaring miRs. Antisense oligonucleotide based siRNA technology using unmodified DNA oligonucleotides are not much effective in miR silencing due to their low-binding affinity (Boutla et al, 2003). But chemically modified single-stranded RNA analogues complementary to specific miRs (ASOs and AMOs) and antagomirs (Valoczi et al, 2004; Jacobsen et al, 2005; Krutzfeldt et al, 2005; Castoldi et al, 2006; Davis et al, 2006; Kloosterman et al, 2006; Naguibneva et al, 2006; Nelson et al, 2006; Orom et al, 2006; Weiler et al, 2006; Busch et al, 2007; Grunweller and Hartmann, 2007) are better option those are widely used in regulating miRNA expression (Esau and Monia, 2007). Wolfrum et al, 2007 have shown that siRNA in conjugation with high-density lipoprotein increase delivery efficacy into only in kidney, gut, liver, and endocrin organs. Along with the delivery method, synthesis and purification of therapeutic grade miR and antagomir is also difficult. Lentivirus mediated and intranasal administration though have been found to be effective in respectively mouse model of breast and lung cancers (Sempere et al, 2007; Yu et al, 2007; Esquela-Kerscher et al, 2008), these methods are required to be standardized to reduce non-targeted site introduction of miR and neuron specific delivery method is yet to be developed (Krutzfeldt et al, 2007). In general, miRs regulate gene expression by complementarity base pairing at 3′ UTRs target mRNAs inhibiting translation or cleaving target mRNAs (Lai, 2002; de Moor et al, 2005; Robins and Press, 2005; Stark et al, 2005; Sun et al, 2005). But truncation mutation of a target gene that removes the miR binding can escape from miR induced repression Therefore, 3′ UTR truncated HMGA2 mediated tumorigenesis (Mayr et al, 2007) may not be controlled by miR therapy.

Table 4. Cardiac myxoma critical disease pathway targeting miRs. miRs are selected on the basis of experimental data and miR analysis databases (mirBase, miRanda, PicTar, TarBase, and TargetScan,)

miRs	Targets
let-7	CCND1-2, CDC25A, CDK4, CDK6, DNA-Polymerases, E2F5-6, FGFR, GRB2, HAND1, HMG2, IGF1, IGFR1, IL6, IL8, MAPK4-6, MMP2, MMP8, MYC, NKX2-5, PCNA, RAS, RB1, TGFB, TGFBR
miR-125	β-catenin, CDC25A, CDK11, DES, E2F3, EDN1/ET-1, ERBB2-4, ESRRA, FGF, FGFR, FMOD, FOS, IGF, IL6R, MAPKs, MIA, MMP11, NFIB, NKX2-5, PCNA, S100A1, SP1, TGFBR1, TNF, VEGF
miR-205	BCL2, CDK4, CDK11, E2F1, E2F5, E2F6, EIF4A3, EIF4E1B, ERBB3, ERBB4, FGF1, FGF4, HRAS, IL5, KRAS, MAPK9, MMP2, NFIB, NOTCH1, S100A1, SP4, VEGFA
miR-214	β-catenin, CCR2, EGFR, HAND1, MUC1, NFKB1, SOX9
miR-217	IL6, PCNA, S100A1
miR-296	CCND1, CCND3, ERBB2, ESR1, FMOD, HAND1, JUN, MMP2, MUC1, NKX2-5, SOX9, TGFB1

IV. Discussion

In this research, using various strategies form bioinformatics and publicly available data, molecular biomarkers in CM have been identified and then a critical disease pathway has been developed using those markers. Later the network is analyzed to identify drug targets. It has been found that cardiac myxomas frequently overexpress VEGF, PCNA, bFGF, FGFR1, IL6, chemokines, MMPs, CALB2, THBD, MUC1, Sox9, Notch1, S100A1, neuron-specific enolase, MIA, and PLA2G2A. Only SM2 is found down-regulated and in very few cases mutations are noticed in P53 and Kras.

Analysis shows that, molecular markers are of diverse functionalities and are involved in heart development, NOTCH signaling, and various growth receptor signaling pathways. Considering all identified markers, when a general critical disease pathway is developed, it has been found that, growth receptor signaling pathways consisting of MYC, FOS, and MMP9 are seems to be the common downstream components of the entire network and CCR2, TGF-β, MUC1, FGFR, EGFR, GATA4, and HAND1 are supposed to be critical key nodes in this network.

Subsequent network analysis reveals that one or more transcription factors, cell cycle regulators, components of replication machinery, tumor suppressor genes, and growth factors are downstream targets of several key nodes (Table 3, Figure 3). For example, PTEN, KRAS, CCND1, ESR1, MMP2, MMP9, and MYC are downstream targets of HER2 and HER2 is regulated by GABPA, MYC, SPI1, AR, EGF, and MUC1. Downstream targets of MYC are found to be FOS, CDC25A, CDK4, eIF4E, and ERBB2. Similarly, PCNA regulates its downstream targets mainly key components of replication machinery such as POLD and E, DNA ligases, and replication factors etc. and itself is regulated by NFkB, E2F4, P53, ESR1, AP1/JUN. Therefore, key nodes, their regulators, and down-stream targets can be considered as drug targets to block that specific pathway unique for certain CM.

There is no clear conclusion about the origin of CM. Though, Johansson in 1989 and Curschellas and colleagues in 1991 suggested an origin of embryonic cell remnants for expression of F8, DES, VIM, Cytokeratins, S100, Lu5, CAM 5.2, and neuron-specific enolase, Terracciano eand colleagues in 2000 predicted an endocardial sensory nerve tissue origin because of CALB2 expression by CM. While report of Farrell and colleagues in 1996 and Lindner and colleagues in 1999 indicates a histogenetic endodermal origin for CM’s positivity for CD34, CD31, CEA, and CA19.9, Kodamaand colleagues in 2002 and Sakamoto and colleagues in 2004 concluded that CM originates from mesenchymal cells and are capable of endothelial differentiation because of their expression of Nkx2.5/Csx, GATA4, cardiac homeobox gene, and eHAND, and no expression of Stem cell factor, Granulocyte colony-stimulating factor, Hepatocyte growth factor, and ET3. In this study, it has been found that common genes those are involved in both heart and myxoma development are Nkx2.5/Csx, GATA4, HOX, HAND, MYOD, SOX4-6, S100, and TGFB. But in case of heart development they mainly act during mesodermal transition at early embryonic stage. As the age group of 0-9 does not show myxoma (Yu et al, 2007), therefore, heart developmental pathway may not be involved rather an epigenetic regulation of these genes may play a role in CM development and further study is required to come to a clear conclusion.

Till now the only effective treatment option is surgical excision of the myxoma though it is costly, risky, and with various post-surgery complaints including recurrences. Again alone chemotherapy is not satisfactory. In search of alternative treatment options, in this study it has been predicted that therapy with a combination of let-7, miR-125, miR-205, miR-214, miR-217, and miR-296 microRNAs those have the potentiality to block the entire critical disease pathway network may be an effective measure in treatment of CM. Very few reports are available regarding the role of miRNAs in cardiac development and cardiovascular disorders. According to van Rooij and colleagues in 2006 miR-23, miR-24, and miR-195 overexpression induces hypertrophic cardiomyocyte growth and upregulation of miR-150 and miR-181b reduces cardiomyocyte cell size. miR-1 is reported to inhibit ventricular cardiomyocyte proliferation and cardiomyocyte differentiation (Zhao et al, 2005) and its induced upregulation prevents hypertrophic growth (Sayed et al, 2007). Table 5 represents a list of heart specific miRs and their cardiac functions and pathophysiology. Among the identified miRs, previous reports shows that let-7 is expressed in cardiac and artery smooth muscles (Lagos-Quintana et al, 2002; Ji et al, 2007) and modulates vascular endothelial cell migration (Kuehbacher et al, 2007; Suarez et al, 2007), miR-125 is expressed in artery smooth muscles (Ji et al, 2007), and over-expression of miR-214 induces hypertrophic cardiomyocyte growth (van Rooij et al, 2006). Except this information, there is no other data available right now regarding the deregulation or specific function of identified miRs in this study in respect to cardiogenesis, cardio vascular disorder, and cardiac myxoma. Therefore, CM specific microRNA expression data is crucial to identify miRs involved in CM pathogenesis and to evaluate the treatment efficacy of these predictive miRs (identified in this research) keeping in mind those difficulties in miR based cancer therapy as discussed earlier. In particular, expression levels in normal and CM condition; there actual roles in cardiogenesis and CM, the delivery methods in situ, and immunogenic and cytotoxic side effects of these miRs should be answered in depth. Similarly, novel targeting agents specific for these identified targets need to be identified and for both these miR and drug based approaches to modulate this complex network of CM pathogenesis require long term experimental studies to identity both the treatment efficacy and possible side effects. Moreover, a systematic, multi-institutional, and worldwide study is essential to make a worldwide database of all prevalent cases and to share these data for in depth study for etiological factors and molecular markers those may help in better understanding

Table 5. Heart specific miRs and their cardiac functions and pathophysiology. p and q indicates respectively up and down-regulation of miR responsible for the pathophysiology.

miRs	Expression	Target genes (predicted/direct)	Cardiac function	Physiopathology	References
let-7	Cardiac and artery smooth muscles		Vascular endothelial cell migration		Kuehbacher et al, 2007 Suarez et al, 2007
miR-1	Heart, skeletal muscle	Rasa1, Cdk9, Rheb, FN1	Heart development, hypertrophy	pCardiac hypertrophy and heart failure	Sayed et al, 2007 Care´ et al, 2007
		HSP60, HSP70	Cardiomyocyte apoptosis		Xu et al, 2007
		CX43, KCNJ2, Kcnn4	Cardiac conduction	pMyocardial infarction, cardiac arrhythmias	Yang et al, 2007
				pDilative cardiomyopathy, coronary artery disease	Xu et al, 2007
	Left ventricle			pEnd-stage heart failure	Thum et al, 2007
				q Dilated cardiomyopathy or aortic stenosis	Ikeda et al, 2007
miR-1-2	Ventricle, skeletal muscle	Gata6, Hrt2, Hand1, Hand2	Cardiac morphogenesis, cardiomyocyte differentiation, inhibits ventricular cardiomyocyte proliferation	qCongenital heart disease, hyperplasia, cardiomyocyte hypertrophy	Zhao et al, 2005
			Cardiomyocyte number		Zhao et al, 2007
		Hand2, Hlf, Rbbp9	Hyperplasia		Zhao et al, 2007
		Irx5, Cx43, IK1, HCN2, HCN4	Electrical conductance	q Arrhythmias and sudden death	Zhao et al, 2007; Yang et al, 2007 Xiao et al, 2007
			Ventricular septation, regulation of heart rate	qLethality due to ventricle wall defect, reduction in heart rate	Zhao et al, 2007
miR-21	Vascular wall, spleen, small intestine, colon	bcl2, PTEN	Vascular smooth muscle cell proliferation and apoptosis, cardiac hypertrophy	pProliferative vascular diseases, cardiomyocyte hypertrophy	Cheng et al, 2007; Sayed et al, 2007 Tatsuguchi et al, 2007 Ji et al, 2007
miR-125	Artery smooth muscles				Ji et al, 2007
miR-133	Heart, skeletal muscle	casp9	Cardiomyocyte apoptosis		Xu et al, 2007
				qTransverse aortic constriction	Van Rooij et al, 2006 Cheng et al, 2007 Thum et al, 2007 Yang et al, 2007
		RhoA, Cdc42, Whsc2	Heart size	qCardiac hypertrophy and heart failure	Care´ et al, 2007
		HERG, HCN2	Electrical conductance	qArrhythmias and sudden death	Xiao et al, 2007 Xiao et al, 2007
miR-195	Heart		Cardiac hypertrophy	qDilated cardiomyopathy, heart failure	Van Rooij et al, 2006
miR-208	Heart	THRAP1	Contraction	q Inhibition of stress induced remodeling, and α-MHC upregulation, cardiac hypertrophy	Van Rooij et al, 2007
miR-214				qHypertrophic cardiomyocyte growth	van Rooij et al, 2006

of the patho-physiology of CM. The most essential thing is the biomarker specific characterization of myxoma cases because the existing evidences show that there are several biomarkers and at least five critical pathways are involved in disease pathogenesis. Identification of unique, case specific, and early stage biomarkers and then subsequent marker specific drug development is important for targeted therapy and to increase treatment efficacy without surgery.

Acknowledgements

We thank to all Bioinformatics software and database providers, whose tools and data were used in this research. We highly appreciate their public, free licensing, academic, and limited trial options.

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From left: Debmalya Barh and Sanjeeb Parida.