Neuregulin-1α and β isoform expression in cardiac microvascular endothelial cells and function in cardiac myocytes in vitro

Abstract

Neuregulins (NRGs) are a family of alternatively spliced growth factors that act through receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family in diverse tissues. The NRG-erbB signaling axis is a critical mediator of cardiac development, and growing evidence supports a role for this system in the intricate cross-talk between the microvascular endothelium and myocytes in the adult heart. The purpose of this study was first to examine the expression of splice variants of the NRG1 gene in adult rat cardiac microvascular endothelial cells and second to compare the function of these variants in cardiac myocytes. We demonstrate that cardiac microvascular endothelial cells in rat culture express multiple Type I NRG1 gene products, including both α and β variants. Comparison of the activity of recombinant NRG1α and NRG1β EGF-like domain proteins in cardiac myocytes shows that the β ligand is a more potent activator of receptor phosphorylation and intracellular signaling than the α ligand, and only the β ligand stimulated glucose uptake and protein synthesis in these culture conditions. Thus, cardiac microvascular endothelial cells express multiple NRG1 isotypes, but only β-variants are biologically active on cardiac myocytes.

Introduction

Neuregulins (NRGs) are a family of growth factors that are ligands for receptor tyrosine kinases in the epidermal growth factor (EGF) receptor family (for review, see [1], [2]). NRGs are made from alternatively spliced transcripts of one of four known genes (NRG1, -2, -3 and -4) and are expressed in diverse tissues. NRGs were originally identified in search of a ligand for the oncogene neu (a.k.a. HER2, erbB2) and have been shown to activate growth, differentiation and survival signaling pathways in multiple cell types including breast epithelial cells, glial cells, neurons, skeletal and cardiac myocytes [3], [4], [5], [6], [7], [8]. In the developing heart, mice deficient in NRG1, or the erbB2 or erbB4 receptors, die in mid-gestation due to a virtually identical malformation of heart trabeculae [9], [10], [11]. In addition, NRG1 and the erbB receptors have been shown to play a role in both murine cardiac conduction system development [12] and heart-valve mesenchyme formation [13].

The importance of the NRG-erbB signaling axis in the adult heart was demonstrated by an unforeseen cardiotoxicity of a novel chemotherapeutic agent targeting the erbB2 receptor: trastuzumab [14], [15]. Characterization of the effects of trastuzumab on cardiac structure and function remains incomplete. At the least, this cardiotoxic effect suggests that alteration of NRG-erbB signaling in the heart can lead to changes in ventricular function, particularly in the setting of myocardial injury. This clinical observation highlights the need for a more complete understanding of the molecular details of NRG-erbB signaling in the heart beyond its role in cardiac development.

Post-natal cardiac myocytes continue to express the erbB2 and erbB4 receptors, but not erbB3, while cardiac microvascular endothelial cells (CMEC) express NRG1 [16]. Neonatal and adult rat ventricular myocytes (NRVM/ARVM) in primary culture respond to recombinant NRG1 II-β3 (rhGGF2) with increased protein synthesis and fetal gene expression [16]. NRG1 II-β3 treatment also improves survival of myocytes through inhibition of apoptotic cell death [16], and rNRG1 EGFβ modulates anthracycline-induced myofilament degradation [17]. These data suggest a role for NRG1β/erbB signaling in the cross-talk between the microvascular endothelium and myocytes in the adult heart that regulates the adaptation of myocardial structure and function to changes in hemodynamic load (see Brutsaert, 2003 for a review of the microvascular endothelium/myocyte relationship [18]).

The NRG1 gene has a complicated structure encoding at least 15 different isoforms from approximately 1.4 megabases of DNA [6], [19]. Distinct NRG isoforms were initially isolated from diverse tissues and with names that reflected their ascribed function including glial growth factor (GGF), acetylcholine-receptor-inducing activity (ARIA), heregulin (derived from ‘regulator of HER2’) and neu differentiation factor (NDF). The defining motif common to all NRG isoforms is an epidermal growth factor-like (EGF) domain that is both necessary and sufficient for activation of receptors (Fig. 1A). The EGF domain is a three-loop structure containing six cysteine residues that form three disulfide bridges. Alternative splicing at the carboxy-terminus of this domain leads to NRGα and β variants, which have distinct affinity for the erbB3 and erbB4 receptors [20]. Depending on the cell and its receptor complement, NRG1β has been reported to be 10–100 times more active than NRG1α [21]. Defects in post-natal breast development are seen in mice lacking NRG1α, but not NRG1β [22], supporting a model where NRGα and β variants serve distinct tissue-specific functions.

The NRG1 gene products can be subdivided into three types (Type I, II and III). Type I and II NRG1 proteins are expressed invariably with a C-2 immunoglobulin-like (Ig) domain that is thought to be involved in the binding of heparan–sulfate proteoglycans in extracellular matrix to increase ligand half-life and thereby activity [23]. In addition, at the amino terminus, Type II NRG1 products include a kringle fold of unknown function. Type III NRG1 are unique in that, rather than expressing an Ig domain, they contain a cysteine-rich domain (CRD). This CRD includes a 5′ hydrophobic sequence that readily associates with the cell membrane. Therefore, unlike Type I and II NRG1, which are type 1 transmembrane proteins that only span the membrane once, it is thought that Type III (CRD) NRG1 has two membrane-spanning regions [24], [25].

Type I NRG1 also express sites for N- and O-linked glycosylation between the Ig and EGF domains (spacer domain). The glycosylation domain is present in nearly all Type I NRG1, but absent from most Type II and all Type III. Immediately adjacent to the transmembrane domain is the stalk domain. Alternative splicing of the stalk domain is responsible for formation of soluble NRG1 (i.e. β3) or one of several full-length transmembrane NRG1 or “pro-NRG” variants (e.g. β1, β2, β4), which are available for proteolytic processing to a soluble ligand. Putative enzymes responsible for stalk cleavage and activation of pro-NRG include members of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, such as tumor necrosis factor α converting enzyme (TACE/ADAM17) [26] or meltrin-β (ADAM19) [27].

Transmembrane NRG1 is expressed with three possible cytoplasmic tail exon combinations (termed “cytoplasmic a, b and c”). Immediately adjacent to the transmembrane region is the common cytoplasmic region, which is followed by exons encoding either the cytoplasmic a or b domains. The cytoplasmic c exon includes an immediate stop codon so the protein is expressed only with the common region. The functions of the cytoplasmic domains remain unknown, but they appear to be required for membrane localization and proteolytic release [28], [29]. Recent evidence suggests that cytoplasmic domains could be involved in “reverse signaling” where the cleaved cytoplasmic tail can migrate to the nucleus as a regulator of transcription [30], [31].

We have previously determined that CMEC express the NRG1 ligand by Northern blot analysis using an extracellular probe common to all Type I and II NRG1 [16]. However, the probe used did not distinguish between the NRG1 isoforms. We therefore developed a strategy to examine specific NRG1 isoforms expressed in CMEC. We found that CMEC express multiple Type I NRG1 including both EGF-like domain α and β splice variants. We compared the effects of recombinant NRG1α and β EGF-like domain proteins (referred to as rNRG1 EGFα and rNRG1 EGFβ, respectively) on cardiac myocytes and found that the β isotype is a more potent activator of erbB receptor phosphorylation and intracellular signaling than α ligand. Moreover, we found that only rNRG1 EGFβ was bioactive in stimulating glucose uptake and protein synthesis in these cardiac myocyte cultures. These data point to a role for NRG1β on target cardiac myocytes, while the function of NRG1α in CMEC remains unknown.