Molecular Cloning of Mouse FK-506 Binding Protein 12.6 Gene and Its Biological Function and Expression Patterns in the Various Models of Heart Disease

摘要

Background and Objectives The FK-506 binding protein 12 (FKBP12) regulates intracellular Ca2+ release by stabilizing the Ca2+-induced Ca2+-release channel (ryanodine receptor) in skeletal muscle. It has been recently shown that a different FKBP, FKBP12.6, is specifically associated with cardiac ryanodine receptor. Since the role of FKBP12.6 in excitation-contraction coupling in the cardiac muscle has not been precisely determined, its biological function was assessed and expression patterns of FKBP12.6 were evaluated in the various models of heart disease. Material and Methods The mouse (m) FKBP12.6 gene was cloned and characterized after screening a mouse genomic DNA library using a mFKBP12.6 cDNA obtained through reverse transcriptase-polymerase chain reaction. Expression levels of mFKBP12.6 was evaluated during cardiac development and in the models of cardiac hypertrophy and failure. Results Both mFKBP12.6 and mFKBP12 contain an open reading frame of 327 nucleotides encoding 108 amino acids. Comparison of mFKBP12.6 cDNA to rat FKBP12.6, human FKBP12.6 and mFKBP12 cDNA revealed 95%, 94% and 74% identity in nucleotide sequence and 98%, 97% and 80% identity in amino acid sequence, respectively. Purified recombinant mFKBP12.6 migrated slower than either mFKBP12 or human FKBP12 on an SDS-polyacrylamide gel, despite having the same number of amino acids and a slightly lower calculated molecular mass. Northern blot analysis showed that the expression of FKBP12 and FKBP12.6 to be highest in brain. While the expression of FKBP12 was much stronger in adult than in embryonic hearts, it was further increased following pressure overload hypertrophy. FKBP12.6 mRNA expression analyzed by RNase protection assay was upregulated after induction of cardiac hypertrophy like FKBP12, whereas it was decreased in the failing heart. The mFKBP12.6 gene contains 5 exons and the proteincoding region of the gene was divided into 4 exon modules. Conclusion We report the molecular cloning and characterization of the mouse FKBP12.6 gene. According to these results, FKBP12 and FKBP12.6 may play a role in the development of cardiac hypertrophy and transition to heart failure. To precisely determine the role of FKBP12 and FKBP12.6 in the heart, a strategy using homologous recombination in embryonic stem cells to conditionally ablate exon 2 of mFKBP12.6 gene has been developed and initial characterization is now underway.