Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes 1,2 . Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle 3 . TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure 4 . Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with α-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres 5 . Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans 6,7 .Forward genetics in zebrafish has led to the identification of several mutations affecting cardiac contractility 8,9 . The most severe and heart-specific of these is sih, which causes a non-contractile heart phenotype ( Fig. 1). Skeletal and smooth-muscle function remain intact in sih mutant embryos, as evidenced by their ability to hatch, swim and show gut peristalsis. One γ-ray-induced allele (sih b109 ) and one chemically induced allele (sih tc300b ) exist 8 . Both of these mutated alleles are fully penetrant and recessive lethal, and sih b109 and sih tc300b mutant embryos are phenotypically indistinguishable. As sih embryos age, pericardial edema develops, the endocardium peels away from the myocardium and the embryos die around seven days post fertilization. Until that time, embryos survive on diffused oxygen and are not dependent on circulating blood 10 .To determine whether the sih phenotype is due to defects in cellular excitation or excitation-contraction coupling, we devised a new assay using the fluorescent calcium indicator Ca 2+ green. In wildtype hearts, a wave of fluorescence representing Ca 2+ influx into cardiomyocytes precedes the contractile wave and progresses from the venous to the arterial end (see Web Movie A and Web Note A online). In mutant hearts, we also observed regular waves of fluorescence, but these were not followed by contraction (Fig. 2a-d). On the basis of this assay, cellular excitation seemed to be intact in mutant cardiomyocytes, indicating that the absence of contractility results from abnormalities downstream of calcium influx.The alignment of thick and thin filaments into sarcomeres creates a highly...
Conventional chemotherapy for cancer has limited specificity for cancer cells. Here, we investigate the possibility of improving the selectivity of chemotherapy by coadministering targeted biological modifier peptides. We show that the 22-amino acid metal-binding transporter domain (MBD) derived from insulin-like growth factor-binding protein-3 selectively targets cancer cells. The rate of MBD uptake by cells was measured using a panel of 54 human cancer cell lines and correlated with MBD cross-linking to cell surface transferrin receptor, caveolin 1, and integrin beta. Gene array data show that MBD uptake correlates with the expression of genes associated with cellular stress-coping mechanisms commonly upregulated in cancer (nuclear factor-kappaB, Hsp-70B). MBD-tagged peptides designed to inhibit such mechanisms have cytotoxic effects on a broad range of human cancer cell lines. The discriminant validity of these peptides as potential cotherapeutic agents was investigated by comparing their cytotoxicity to cancer cell lines versus normal human cell counterparts. Synergies between these peptides and marginally cytotoxic levels of 5-fluorouracil were demonstrated. Biodistribution data from in-vivo experiments in mice and rats confirm that MBD-tagged peptides and proteins preferably localize to specific tissues, such as kidney and pancreas. Intracardial injection of CCRF-CEM T-cell leukemia or MDA-MB-435 cells into Rag-2 mice establishes disseminated disease within 7 days. Twenty-five-day subcutaneous administration of a three-peptide cocktail (3 mg/kg) in combination with 5-fluorouracil in Rag-2 mice with established CCRF-CEM leukemia significantly reduces splenomegaly and bone marrow cancer cell burden. In a similar experiment using MDA-MB-435 cells, MBD-tagged peptides reduced human cell burden in bone marrow. Taken together, these data suggest that MBD-tagged molecules can be used as highly selective chemosensitizers in the treatment of hematological and disseminated malignancies.
The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.
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