No abstract

The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenous Adss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and these cis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2.The murine linear heart tube begins to contract spontaneously by 8 d.p.c. 1 (1) and pumps continually throughout the lifetime of the animal; therefore, the metabolic pathways that provide energy must be properly activated and regulated. Several genes that encode metabolic enzymes are activated at low levels during early cardiogenesis, then are up-regulated around birth to reach adult levels during postnatal development to deal with the increasing cardiac energy demands (2, 3). Numerous metabolic pathways also utilize cardiac-specific isozymes that are present at high levels in the heart (4 -6). Although the biochemistry of cardiac metabolism is well defined, the genetic signaling pathways that control expression of the cardiac metabolic genes are not well understood. Few cardiac cis-regulatory elements that function in vivo have been identified and preliminary studies are just beginning to describe transcription factor interactions as genetic regulatory mechanisms. The subset of cardiac regulatory regions that has received the most attention has been those that control contractile gene transcription (7-9). Two of the best characterized cardiac structural gene enhancer and promoter regions regulate the ␣-and ␤-myosin heavy chain genes, which have been used to direct expression of several other genes to the murine heart (10 -12). Less well characterized cardiac structural gene enhancers include those that control expression of the myosin light chain 1/3 (13) and 2v genes (14). Another class of cardiac elements, those regulating genes that encode enzymes of energy metabolism, is the least understood group of cardia...

A muscle-specific isoform of adenylosuccinate synthetase (AdSS1, EC 6.3.4.4) is one of three enzymes that constitute the purine nucleotide cycle, a muscle-specific metabolic cycle. Previously, we showed that the muscle Adss1 gene was highly expressed in both skeletal muscle and heart of the adult mouse. Here we have shown that the Adss1 gene is initially activated early in embryonic development in skeletal muscle and heart precursors and is subsequently up-regulated perinatally. The earliest detectable gene expression corresponds with the establishment of the first myogenic and cardiac lineages. To allow identification of the genetic signals controlling this developmental pattern of expression, the Adss1 gene was cloned and its structure determined. Transgenic analysis has shown that 1.9 kilobase pairs of 5 flank can activate expression in skeletal muscle progenitors and direct enhanced expression to adult cardiac muscle. Sequence analysis of the promoter and 5 flanking region revealed the presence of numerous potential muscle-specific cis-regulatory elements.Studies aimed at understanding muscle gene regulation have traditionally focused on three classes of tissue-specific genes, those encoding myogenic determination factors, those encoding contractile proteins, and those encoding enzymes of energy metabolism (1, 2). The myogenic transcription factors are instrumental in all phases of myogenesis (commitment, differentiation, and maturation) and as such are expressed in a very tightly controlled temporal fashion throughout skeletal muscle and cardiac development (3, 4). Members of the second class, the contractile proteins, are considered the building blocks of muscle and cardiac fibers and accordingly do not accumulate until the onset of myotube formation during the differentiation process (5). Unlike the myogenic factors, these structural proteins (specifically the actins and myosins) progressively accumulate throughout the prenatal and postnatal phases of development, some actually reaching levels as high as 30% of adult levels at birth. Several embryonic and/or fetal isoforms disappear around the time of birth, as the levels of the adult isoforms increase during the initial period of postnatal development. The third class of muscle-specific genes encodes enzymes of muscle energy metabolism. These genes are not highly expressed until late in embryogenesis (i.e. right before birth), after which the genes are highly up-regulated (20 -30-fold) during the first weeks of postnatal development (6). Consistently, their cognate enzymes are abundant in adult cardiac and skeletal muscle. For several of these cardiac and skeletal muscle metabolic genes, it has been demonstrated that musclespecific transcripts can be detected at low levels early in development (7-12). In fact, earliest detection of muscle-specific transcripts for the ␤-enolase gene corresponds with the presence of the first muscle progenitors and the primordial cardiac tube (8). This suggests that the increased expression of the muscle metabolic genes du...