Expression and functional activity of four myocardin isoforms

Abstract

Myocardin (MYOCD) is an essential component of a molecular switch for the expression of contractile genes in smooth muscle and cardiac muscle cells. The Myocd gene comprises at least fifteen exons, including two alternately spliced exons designated 2a and 10a. We investigated tissue-specific Myocd expression in mouse, rat and human tissues to determine the conservation in expression of each Myocd splice variant and to ascertain whether any functional differences exist among MYOCD isoforms. Conventional and quantitative RT-PCR revealed the dominant expression of Myocd exon 2a (Myocd_v3) in smooth muscle cell (SMC)-rich tissues (aorta and bladder) with little expression in heart across all species studied. Each species of heart showed primarily a longer version of Myocd (Myocd_v1) without exon 2a. While exclusion of exon 2a was common in all cardiac muscle samples, exon skipping of Myocd exon 10a was a rare event in both cardiac muscle and SMC tissues. In general, all four MYOCD isoforms showed comparable stimulation of SMC promoters. On the other hand, Myocd_v1 and Myocd_v2 were more active than Myocd_v3 and Myocd_v4 in stimulating cardiac muscle promoters and Myocd_v1's activity was augmented in the presence of the cardiac transcription factor, MEF2C. Importantly, whereas all four MYOCD isoforms similarly induced expression of endogenous SMC genes in a prostate tumor cell line (LNCaP), none could induce the endogenous expression of specific cardiac markers. These results are the first to show relative expression and activities of the major myocardin isoforms across disparate species. We propose a new myocardin nomenclature reflecting the dominant splice variants expressed in cardiac muscle (Myocd_v1 and v2) versus SMC-rich tissues (Myocd_v3 and v4).

Introduction

Myocardin (MYOCD) is a crucial component of a molecular switch for the induction of contractile gene expression in smooth muscle cells (SMCs) and cardiac muscle cells (Wang et al., 2001, Chen et al., 2002, Wang et al., 2003). MYOCD mediates its activity primarily through direct association with serum response factor (SRF), which binds CArG elements located in the regulatory regions of many SMC structural genes (Miano, 2003). In addition, MYOCD and SRF can activate key SMC regulatory genes such as myosin light chain kinase and the beta 1 subunit of the calcium-activated potassium channel (Wang et al., 2003, Zhou and Herring, 2005, Yin et al., 2006, Long et al., 2009). Altered expression of Myocd mRNA disturbs homeostasis of the SMC contractile gene program in vitro and in vivo (Chen et al., 2002, Hendrix et al., 2005, Tharp et al., 2006, Chow et al., 2007). Ectopic expression of Myocd reconstitutes the SMC contractile gene program in vitro and is sufficient for the biochemical, ultrastructural, and physiological states of these cells (Long et al., 2008). Moreover, genetic ablation of SMC impairs vascular SMC differentiation and normal function (Li et al., 2003, Huang et al., 2008). Thus, MYOCD is the main transcriptional switch governing SMC differentiation.

The mouse Myocd gene has fifteen exons, including two exons that arise from alternative splicing. Exon 2a of Myocd is reported to be expressed specifically in SMCs and is characterized by the presence of an in-frame stop codon, which leads to alternative translation from another start codon located further downstream at amino acid position 79 within exon 4 (Creemers et al., 2006). Exon 10a was initially described as a cardiac muscle-specific transcript but was shown subsequently in SMCs as well (Ueyama et al., 2003, van Tuyn et al., 2005, Torrado et al., 2009). While this work was in progress, another group reported additional, rare alternate exons between exons 2 and 3 (Saha et al., 2009).

A paucity of information exists about the relative expression of each Myocd isoform across tissues in multiple species. Further, essentially nothing is known about the relative activity of each MYOCD isoform. In this report, we provide an analysis of Myocd isoform expression in cultured cells and tissues from mouse, rat and human. We find conserved tissue-specific patterns of expression for each Myocd isoform and go on to show MYOCD isoform-specific activities directing SMC versus cardiac muscle gene expression. We propose the use of HUGO-approved nomenclature for the four dominant Myocd splice variants that exist in nature.