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The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

ABSTRACT

Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.

Results: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.

Conclusion: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

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Expression of β2 subunit transcript variants in the embryo and adult. RT-PCR analysis using transcript variant-specific primers (located in the 5' exons 1 or 2 and exon 10) was performed on RNA samples from A) whole embryos at various developmental stages, B) cardiac tissue dissected from cmlc2:GFP embryos or from adult fish, and C) adult organs and tissues. Expression of a housekeeping gene, EF1α, was used as a control for RNA integrity. In B, 72 hpf or adult RNA reactions were run on single gels, subsequently subdivided to multiple panels for clarity in presentation. Transcript variant numbers are listed to the right of panels; refer to Fig. 1D.
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Figure 7: Expression of β2 subunit transcript variants in the embryo and adult. RT-PCR analysis using transcript variant-specific primers (located in the 5' exons 1 or 2 and exon 10) was performed on RNA samples from A) whole embryos at various developmental stages, B) cardiac tissue dissected from cmlc2:GFP embryos or from adult fish, and C) adult organs and tissues. Expression of a housekeeping gene, EF1α, was used as a control for RNA integrity. In B, 72 hpf or adult RNA reactions were run on single gels, subsequently subdivided to multiple panels for clarity in presentation. Transcript variant numbers are listed to the right of panels; refer to Fig. 1D.

Mentions: To determine whether β2 genes are expressed in a stage- or tissue-specific manner in early embryogenesis, we performed RT-PCR on RNA isolated from embryos of several stages in early development. To track the expression patterns of specific transcript variants (Fig. 2D), we used forward primers specific to the 5' exons 1 or 2, and reverse primers in exon 10 (the GK domain) in RT-PCR experiments (Fig. 7). Surprisingly, amplification of β2.1 and β2.2 transcripts occurred in embryos as young as the 4-cell and 1000-cell stages (Fig. 7A). Since zebrafish zygotic transcription does not initiate until the 10th cell division (~the 1000-cell stage) [56], the presence of mRNA in 4-cell embryos indicates that the transcripts are of maternal origin. The β2.2 transcript variant 2 was expressed steadily from the 4-cell stage through 72 hours post-fertilization (hpf). In contrast, β2.2 transcript variant 1 showed a pulse of expression in early epiboly stages. β2.1 transcript variant 6 was robustly detected from 26 hpf through at least 3 dpf. Other β2.1 transcript variants (1 and 2) were detected more sporadically or were undetectable in this assay, consistent with their rare recovery in RACE reactions. Thus, both β2.1 and β2.2 are expressed from the earliest stages of embryogenesis, but show significant heterogeneity in patterns of transcript variant expression throughout the first three days of embryogenesis.


The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

Expression of β2 subunit transcript variants in the embryo and adult. RT-PCR analysis using transcript variant-specific primers (located in the 5' exons 1 or 2 and exon 10) was performed on RNA samples from A) whole embryos at various developmental stages, B) cardiac tissue dissected from cmlc2:GFP embryos or from adult fish, and C) adult organs and tissues. Expression of a housekeeping gene, EF1α, was used as a control for RNA integrity. In B, 72 hpf or adult RNA reactions were run on single gels, subsequently subdivided to multiple panels for clarity in presentation. Transcript variant numbers are listed to the right of panels; refer to Fig. 1D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365960&req=5

Figure 7: Expression of β2 subunit transcript variants in the embryo and adult. RT-PCR analysis using transcript variant-specific primers (located in the 5' exons 1 or 2 and exon 10) was performed on RNA samples from A) whole embryos at various developmental stages, B) cardiac tissue dissected from cmlc2:GFP embryos or from adult fish, and C) adult organs and tissues. Expression of a housekeeping gene, EF1α, was used as a control for RNA integrity. In B, 72 hpf or adult RNA reactions were run on single gels, subsequently subdivided to multiple panels for clarity in presentation. Transcript variant numbers are listed to the right of panels; refer to Fig. 1D.
Mentions: To determine whether β2 genes are expressed in a stage- or tissue-specific manner in early embryogenesis, we performed RT-PCR on RNA isolated from embryos of several stages in early development. To track the expression patterns of specific transcript variants (Fig. 2D), we used forward primers specific to the 5' exons 1 or 2, and reverse primers in exon 10 (the GK domain) in RT-PCR experiments (Fig. 7). Surprisingly, amplification of β2.1 and β2.2 transcripts occurred in embryos as young as the 4-cell and 1000-cell stages (Fig. 7A). Since zebrafish zygotic transcription does not initiate until the 10th cell division (~the 1000-cell stage) [56], the presence of mRNA in 4-cell embryos indicates that the transcripts are of maternal origin. The β2.2 transcript variant 2 was expressed steadily from the 4-cell stage through 72 hours post-fertilization (hpf). In contrast, β2.2 transcript variant 1 showed a pulse of expression in early epiboly stages. β2.1 transcript variant 6 was robustly detected from 26 hpf through at least 3 dpf. Other β2.1 transcript variants (1 and 2) were detected more sporadically or were undetectable in this assay, consistent with their rare recovery in RACE reactions. Thus, both β2.1 and β2.2 are expressed from the earliest stages of embryogenesis, but show significant heterogeneity in patterns of transcript variant expression throughout the first three days of embryogenesis.

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

ABSTRACT

Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.

Results: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.

Conclusion: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Show MeSH
Related in: MedlinePlus