No abstract

To understand how to reinitiate cell division in adult human myocardium, a heart regeneration model was examined in the amphibian axolotl salamander, Amblystoma mexicanum. The ventricular apex was surgically amputated and resected for 3 weeks. At 14 days of recovery, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was injected intraperitoneally to identify cell types undergoing S-phase by indirect immunofluorescence using primary anti-BrdU antibodies. This is the first report showing a concentrated area of cell division in the ventricle and cells throughout the atrial epicardium by confocal microscopic image analysis in response to wounding of the ventricle. Tissues coimmunostained with anti-BrdU and sarcomeric myosin-specific MF20 antibodies showed 12.8 +/- 4.10% of myocytes contained BrdU(+) nuclei in a 75 microm to 750 microm zone in the ventricular myocardium subjacent to the amputation plane. BrdU(+) nuclei also were present in newly formed ventricular epicardium that surround dividing myocytes, and in epicardial mesothelium (74.3 +/- 6.36 %) and connective tissue (44.9 +/- 13.31%) cells distal to the wound. Unexpectedly, immunofluorescent BrdU(+) nuclei were observed in isolated atrial myocytes (13.9 +/- 1.45%) and in uninjured atrial epicardial mesothelium (64.3 +/- 1.55%) and connective tissue (29.4 +/- 5.50%). No BrdU(+) nuclei were present in cardiomyocytes or epicardium from sham-operated and BrdU-treated controls. These results suggest that renewed cell division is a specific response to wounding of the ventricle. Additionally, release of a growth factor may be responsible for the specific localized mitotic ventricular cardiomyocyte response adjacent to the wound, and the more generalized atrial proliferative response distal to the amputation.

Antibodies directed against thyroxine-binding globulin (TBG) have been used to screen a human liver Xgtll expression library. A 1.46-kilobase clone was identified which encodes nearly the complete amino acid sequence, beginning at amino acid 17 of the mature protein. To complete the protein sequence, the cDNA clone was used to identify a genomic clone coding for TBG in a human X chromosome library. The overlapping recombinant clones contained an open reading frame coding for 415 amino acids followed by a polyadenylylation signal (AATAAA) located 275 nucleotides from a TAG termination codon. Beginning at residue 21, the deduced amino acid sequence agrees closely with the known NH2-terminal sequence of the mature peptide. The preceding 20 amino acid residues are hydrophobic in character and presumably represent a leader sequence. Four glycosylation sites were identified, corresponding to the number determined for the purified protein. DNA blot hybridization revealed a single-copy gene, which by chromosomal analysis was found to be located on the long arm of the X chromosome. Unexpectedly, the nucleotide sequence of TBG is closely homologous to those encoding the plasma serine antiproteases a1-antichymotrypsin and a1-antitrypsin. However, there is little overall homology between TBG and transthyretin (prealbumin), the other major thyroxine-binding protein of human plasma.In most vertebrate species, thyroxine-binding globulin (TBG) has been reported to be the major thyroxine-binding protein (1). It is a glycoprotein (molecular mass about 54 kDa) that is synthesized in the liver as a polypeptide of 45 kDa (2). TBG represents a relatively rare translation product of total liver mRNA, accounting for only about 0.018% of total acidprecipitable radioactivity (2). Efforts to obtain detailed information about the structure of TBG and its thyroxinebinding site have been hampered by the presence of multiple electrophoretic forms and the inability to crystallize the protein (3, 4).In this report, we describe the use of serum containing polyclonal antibodies directed against human TBG to isolate cDNA clones coding for TBG from a human liver Xgtll expression library (5). To complete the coding sequence, one of these clones was used to identify a genomic clone from a human X chromosome library. Residues 21-40 of the deduced amino acid sequence correspond closely to the NH2-terminal amino acid sequence of mature TBG reported by Cheng (6). Amino acids 1-20 are very hydrophobic in nature and presumably represent the leader sequence of the propeptide. Unexpectedly, TBG shows a high degree of homology to a1-antichymotrypsin and a1-antitrypsin, and appears to belong to the serpin superfamily, most of which are serine proteinase inhibitors (7,8

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin beta1 and correlate with changes that occur in beta1 spliced variants and their respective alpha isoforms.