Hypoplastic left heart syndrome myocytes are differentiated but possess a unique phenotype

Abstract

Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available. Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry. Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or “heart failure” gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n=21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.

Introduction

The term hypoplastic left heart syndrome (HLHS) is used to describe a group of malformations characterized by marked underdevelopment of the left side of the heart. The anatomic abnormalities include underdevelopment of the left atrium and ventricle, stenosis or atresia of the aortic or mitral orifices, and marked hypoplasia of the ascending aorta [1], [2]. HLHS accounts for approximately 7–8% of heart disease producing symptoms in the first year of life and is the most common cause of death from heart disease in the first week after birth [3]. It accounts for nearly 25% of cardiac deaths in the first year of life (New England Regional Infant Cardiac Registry). The prognosis for most patients with HLHS is bleak and treatment is surgery to repair the defects or, more rarely, cardiac transplantation. HLHS is unlike most other complex congenital heart diseases in that abnormalities in other organ systems are rare. Thus, children with HLHS are, except for heart abnormalities, normal [4]. The genetic basis of HLHS is not clear, although in some cases the syndrome has been reported to be associated with chromosomal abnormalities [5], [6].

Microscopic examination of tissue sections from both right and left ventricles of patients with HLHS shows disorganized, variably sized bundles of myocytes coursing through abundant loose connective tissue and scattered, dilated, thin-walled blood vessels. Myocytes are focally small with scant cytoplasm. It is not known if HLHS myocytes express proteins typical of differentiated myocytes or remain significantly undifferentiated.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily [7]. The 130-kDa full-length protein possesses six extracellular immunoglobulin domains, a transmembrane portion and a cytoplasmic domain [8]. Functionally, PECAM-1 is an adhesion molecule with both homophilic and heterophilic binding. The homotypic binding is important in leukocyte transendothelial migration [9]. The heterotypic ligands have been reported to include integrin, αvβ3 [10] and glycosaminoglycans [11]. PECAM-1 is present on the surface of platelets, some white blood cells and endothelial cells [7], [12], and is strongly expressed by all endothelial cells and to a lesser extent on several types of leukocytes [13]. Immunohistochemical staining for PECAM-1 is routinely used to demonstrate angiogenesis in several types of cancer [14], [15], [16], [17], [18], [19]. Protein expression studies have demonstrated that PECAM-1 is leukocyte and endothelial cell-specific and that PECAM-1 is not expressed in mouse cardiac myocytes at any time during development or in any cardiac diseases [20], [21], [22]. However, Hwang et al. reported expression of PECAM-1 in 8–12-week fetal human hearts but not in older fetal or adult hearts. Since this analysis was cDNA-based, it cannot be determined if PECAM-1 mRNA expression in fetal hearts of this age is localized to myocytes or other cell types within the myocardium [23]. In addition, PECAM-1 null mice have no discernible cardiovascular malformations and therefore PECAM-1 expression is not necessary for normal cardiovascular development in the mouse [24].

With improvements in early diagnosis of HLHS and the increasing potential for surgical intervention in the fetus, it is essential to determine the pathophysiological basis for the ventricular malformations and to determine the potential for HLHS myocytes to respond to normalized ventricular flow. If the ventricular myocytes are unable to respond appropriately following surgical correction, early intervention may be futile. We have used immunohistochemistry, quantitative reverse transcription polymerase chain reaction (QRTPCR) and two-dimensional (2-D) gel electrophoresis coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to determine that HLHS left ventricular myocytes have a differentiated but unique phenotype. The myocytes express mRNAs and proteins characteristic of differentiated, failing cardiac myocytes and form striations and intercalated discs. However, HLHS myocytes in all patient samples (n=21) inappropriately express PECAM-1, a member of the CAM family that has a primary role in the regulation of tissue morphogenesis. Cardiac myocytes have never been shown to express PECAM-1 at any stage of development or in any disease state. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.