Abstract
Calsequestrin is the main calcium-binding protein inside the sarcoplasmic reticulum of striated muscle. In mammals, the cardiac calsequestrin gene (casq2) mainly expresses in cardiac muscle and to a minor extent in slow-twitch skeletal muscle and it is not expressed in non-muscle tissues. This work is the first study on the transcriptional regulation of the casq2 gene in cardiac and skeletal muscle cells. The sequence of the casq2 genes proximal promoter (180 bp) of mammals and avians is highly conserved and contains one TATA box, one CArG box, one E-box, and one myocyte enhancer factor 2 (MEF-2) site. We cloned the human casq2 gene 5'-regulatory region into a luciferase reporter expression vector. By functional assays we showed that a construct containing the first 288 bp of promoter was up-regulated during myogenic differentiation of Sol8 cells and had higher transcriptional activity compared with longer constructs. In neonatal rat cardiac myocytes, the larger construct containing 3.2 kb showed the highest transcriptional activity, demonstrating that the first 288 bp are sufficient to confer muscle specificity, whereas distal sequences may act as a cardiac-specific enhancer. Electrophoretic mobility shift assay studies demonstrated that the proximal MEF-2 and CArG box sequences were capable of binding MEF-2 and serum response factor, respectively, whereas the E-box did not show binding properties. Functional studies demonstrated that site-directed mutagenesis of the proximal MEF-2 and CArG box sites significantly decreased the transcription of the gene in cardiac and skeletal muscle cells, indicating that they are important to drive cardiac and skeletal muscle-specific transcription of the casq2 gene.
Highlights
Intracellular calcium, storing Ca2ϩ up to 20 mM while maintaining the free SR Ca2ϩ concentration at ϳ1 mM [2, 3]
The analyses revealed that both clones overlap and contain the complete casq2 gene, integrated with 11 exons and interrupted by 10 introns that span over 68 kb of the genome (Fig. 1A)
Calsequestrin has a major role to maintain the calcium homeostasis in the striated muscle cells, the data regarding its specific tissue expression and absence of changes on its expression associated with pathological conditions, evidences the existence of a fine regulation of its expression
Summary
CASQ2 is the predominant isoform during the fetal period, and during neonatal life. Afterward, there is a switch to CASQ1 gene expression, making the CASQ1 isoform the only one found in adult muscle. In transgenic mice that overexpress CASQ2, it was observed that the mice develop cardiac hypertrophy and heart failure, with a typical fetal phenotype, associated with a higher Ca2ϩ storage capacity of the SR, as well as an impaired Ca2ϩ release, which leads to a diminished contractility [11] Another pathological state where CASQ2 is involved is the catecholaminergic polymorphic ventricular tachycardia (CPVT), where several mutations in the casq gene are present, inserting stop codons in the first exon of the gene, nullifying the expression of the casq gene. The results obtained in this work suggest that the proximal promoter is necessary and sufficient for cardiac and skeletal muscle expression, and that MEF-2 and SRF transcription factors participate for the tissuespecific expression of the casq gene. 5Ј-TGCATGATTTATTTTTAGCCTGAAACAAC-3Ј 5Ј-GATCGCTCTAAAAATAACCCTGTCG-3Ј 5Ј-CATTGCATGCTGGATCCTCCGCCTGAAACAAC-3Ј 5Ј-GATCGCTGTAAACATAACCCTGTCG-3Ј 5Ј-CTGCATCCTAAAAATGGAGTTCC-3Ј 5Ј-GGATGTCCATATTAGGACATCT-3Ј 5Ј-CTGCATCCTAAAAATTTAGTTCC-3Ј 5Ј-GGATGTCCATATTATTACATCT-3Ј
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