Atrial Chamber-specific Expression of the Slow Myosin Heavy Chain 3 Gene in the Embryonic Heart

Abstract

The quail slow myosin heavy chain 3 (slow MyHC 3) gene is expressed in the developing heart and in slow muscles of the developing limb. It is first expressed in the pulsatile cardiac tube in the embryo, and as the heart chamberizes its expression becomes restricted to the atria. To identify regulatory elements responsible for atrial-specific expression, the 5 upstream region of slow MyHC 3 gene was investigated. An atrial regulatory domain (ARD1) between ؊840 and ؊680 acts as an atrial cell-specific enhancer in primary cardiocyte cultures. ARD1 also specifies atrial-specific expression in vivo when the ARD1/heterologous promoter was introduced into developing chick embryos by a replication-competent retroviral vector. ARD1 is the first atrial cell-specific enhancer to be identified. Fine deletion and mutation analysis within ARD1 defined a 40-base pair vitamin D 3 receptor-like element that controls atrial cell-specific expression of the slow MyHC 3 gene by inhibiting its expression in ventricular cardiocytes.

A Positive GATA Element and a Negative Vitamin D Receptor-Like Element Control Atrial Chamber-Specific Expression of a Slow Myosin Heavy-Chain Gene during Cardiac Morphogenesis

“…AMHC1 is first expressed in the posterior region of the fusing chicken heart tube, the future atrial compartment, by stage 9 (60). A 160-bp region of the slow MyHC 3 gene promoter, designated ARD1, has been shown to act as an atrial chamber-specific enhancer both in cell culture and in the embryo (56). A vitamin D receptor like sequence motif present within the enhancer is required for the observed inhibition of reporter expression in ventricular but not in atrial cardiomyocytes, supporting the hypothesis that atrial chamber-specific expression is achieved by ventricular chamber-specific inhibitors (56).…”

“…MyHC immunostaining of cultured cardiomyocytes with monoclonal antibodies F59 and S58 was performed as described previously (56). Monoclonal antibody F59 recognizes all known avian fast MyHC isoforms (6), while in the avian heart, S58 is specific for the slow MyHC 3 isoform (56).…”

“…The myosin heavy-chain (MyHC) AMHC1 and slow MyHC 3 genes (56,60), the myosin light-chain 1a (MLC-1a) and MLC-2a genes (21,31), and the atrial natriuretic factor (ANF) gene (61) show atrial specificity. In small mammals, the ␣-MyHC gene exhibits an atrial chamber preference of expression before birth (31).…”

Identification and Genomic Cloning of CMHC1

“…The quail slow myosin heavy chain 3 gene is expressed throughout the heart until embryonic day 7 when ventricular expression is repressed. Expression of quail slow MHC 3 is also detected in embryonic slow skeletal muscles (14,15). VMHC1 expression is detected throughout the developing chick heart until day 5 of development when its cardiac expression is restricted to the ventricle.…”

“…Tissue Culture and in Vitro Transfections-Primary cardiomyocytes were isolated from hearts of embryonic day 11 White Leghorn chickens (Truslow Farms) as described previously (15). NIH 3T3 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium ϩ 20% fetal bovine serum.…”