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Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart
A Greig1, Y Hirschberg, P A Anderson
1Department of Biology, University of North Carolina at Chapel Hill 27599.
Abstract:
In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Rabbit cardiac troponin T (cTnT) diversity arises from alternative splicing of three nucleotide sequences within the gene. This molecular basis explains the different cTnT isoforms and their impact on heart muscle calcium sensitivity.
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Protein Isoforms
Background:
- Multiple cardiac troponin T (cTnT) isoforms exist in the rabbit heart, correlating with myofilament calcium sensitivity.
- The molecular basis for this cTnT diversity remained to be elucidated.
Purpose of the Study:
- To establish the molecular basis for the diversity of cardiac troponin T (cTnT) isoforms in the rabbit heart.
- To investigate the role of alternative splicing in generating different cTnT protein variants.
Main Methods:
- Restriction-digest analysis of rabbit genomic DNA to determine cTnT gene copy number.
- Isolation and sequencing of cTnT cDNA clones to establish a consensus protein sequence.
- Reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of newborn rabbit heart cDNAs to identify and analyze variant sequences.
- In vitro transcription and translation of cDNA clones to generate and characterize protein isoforms.
Main Results:
- The rabbit cTnT gene is a single-copy gene.
- Alternative splicing involving the variable inclusion/exclusion of an 18-nt and a 30-nt sequence in the 5' half of the cDNAs generates multiple cTnT products.
- A 9-nt region in the 3' half of the cDNAs, homologous to rat cTnT exon 12, also exhibits heterogeneity.
- In vitro studies confirmed that specific combinations of these variable sequences generate protein isoforms corresponding to known cTnT variants (cTnT1-cTnT4).
Conclusions:
- Alternative splicing of specific nucleotide sequences within the cardiac troponin T (cTnT) gene is the primary mechanism generating diverse cTnT isoforms in the rabbit heart.
- These splicing events contribute to the observed variations in myofilament calcium sensitivity.
- The findings provide a molecular explanation for cTnT isoform diversity and its functional implications in cardiac muscle.