Serum troponin T: diagnostic marker for acute myocarditis.
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Acute Disease↗
Biomedical subjects
W M Franz
Publications and source records attributed to W M Franz.
Heart muscle-specific gene expression using replication defective recombinant adenovirus.
Adenoviruses are a promising vector system for future gene therapy of heart muscle diseases. The promiscuous tissue tropism of adenoviruses, however, may lead to the undesirable expression of putative therapeutic genes in nontarget cells and hence to considerable safety limitations for this vector system. To restrict gene expression to cardiomyocytes we constructed an adenoviral vector (Ad-mlcLuc) in which the luciferase gene is under the control of the ventricle-specific myosin light chain-2 (mlc-2v) promoter. For controls, we constructed a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (RSV) promoter (Ad-rsvLuc). Our data demonstrate that the newly established viral vector Ad-mlcLuc was specifically active in rat neonatal cardiomyocytes in vitro but not in three established cell lines. Injections of the recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in myocardial specific gene expression of Ad-mlcLuc in vivo, despite the fact that viral DNA was detected by PCR at different levels in all tissues investigated. In vitro and in vivo, Ad-mlcLuc was exclusively active in cardiac muscle cells, reaching 8-9% of the RSV-induced luciferase activity. Direct injection of Ad-mlcLuc into thigh muscle gave only background luciferase activity (0.05% of Ad-rsvLuc). Therefore, in the adenoviral system, the mlc-2v promoter allows heart-specific expression of a foreign gene thus providing a promising tool for gene transfer targeted to the myocardium.
Adenoviruses, Human↗
X-linked dilated cardiomyopathy. Novel mutation of the dystrophin gene.
We report on a family with a severe form of X-linked dilated cardiomyopathy (DCM). Two brothers, the elder requiring heart transplantation, and a maternal cousin presented elevated creatine kinase levels, increased right ventricular diameters and electrocardiographic abnormalities. All complained of exertional cramping myalgia, but none had muscle weakness or a pathological electromyogram. Muscle biopsies of these individuals revealed a mild myopathic picture with atrophic type I and hypertrophic type II fibers. Immunofluorescence using N- and C-terminal antibodies (dys-2, dys-3) against the dystrophin protein showed preserved, but reduced intensity of staining of the sarcolemmal membranes. Using the same two antibodies, Western blot analyses revealed a dystrophin molecule of the expected molecular weight, which was quantitatively reduced by 80%. However, the dys-1 antibody, directed against the mid rod region of the dystrophin protein, did not react with dystrophin both on Western blot and immunofluorescence. Linkage analysis with polymorphic markers of the dystrophin gene revealed an identical haplotype at the 5' region in all affected individuals (two point lod score of 1.93, phi = 0). A deletion of exons 48, 45-53, 2-7 and 1 including the promoter region of the dystrophin gene, as described in rare cases with similar clinical signs could be excluded by multiplex PCR and Southern blot analyses of this DCM family. In addition, a major splice-mutation of dystrophin mRNA was excluded by RT-PCR of skeletal and heart muscle tissue. Therefore, we conclude that a novel mutation in the 5' region of the dystrophin gene phenotypically leads to this severe form of DCM. Extensive analyses of the dystrophin gene, in particular of the sequences coding for the antigenic determinants of the dys-1 antibody in the mid rod region, may identify the molecular cause of this monogenetic form of DCM.
Adult↗
[A transgenic animal model: new possibilities for cardiovascular research].
By means of molecular biology and genetic research the influence of genetic factors on a great variety of human diseases could be shown. In the field of cardiovascular research the genetic defects of a few monogenetic disorders, such as Marfan's syndrome and hypertrophic cardiomyopathy, have been characterized. In addition, candidate genes for polygenetic diseases, such as arterial hypertension and atherosclerosis, have been cloned. However, the identification of a candidate gene or its mutation does not prove its influence on the phenotype or the final cause of a particular disease. Only a targeted manipulation of a defined candidate gene in a transgenic animal model helps to understand the role of the gene and its product in the whole organism. Transgenic experiments can be divided into gene-addition and gene-deletion models. In a gene-addition experiment a fusiongene is microinjected into a fertilized oocyte. The fusiongene itself consists at least of a regulatory element promoter and of a DNA sequence coding for the gene product (protein) of intended overproduction. The choice of the right promoter is important for obtaining tissue-specific gene expression. The cardiac myosin light chain-2 promoter for example leads to a ventricle-specific gene expression in cardiomyocytes from early embryogenesis through adulthood. In a gene-deletion experiment on the other hand, the target gene is selectively knocked out by homologous recombination in embryonic stem (ES) cells. The selected ES-cells are then injected into blastocysts. If the ES-cells are integrated into the germ line and transmitted to the progeny, a transgenic line is established. This review article describes planning and development of transgenic animals and discusses established transgenic animal model systems with regard to cardiovascular physiology. In addition, animal models which may provide a basis for future gene therapy will be introduced.
Animals↗
Characterization of a cardiac-selective and developmentally upregulated promoter in transgenic mice.
Transcriptional regulatory mechanisms which mediate cardiac-specific gene expression have not yet been completely understood. Potential cardiac-specific promoter sequences, sharing similar protein binding motives, show either coexpression in skeletal muscle, local restriction to the atrium or late onset of expression during fetogenesis. Based on in situ hybridization studies that indicated the expression of the cardiac myosin-light-chain-2 (MLC-2) gene in ventricular myocardium and in the lower outflow tract, a model system for selective targeting of foreign genes to the heart of transgenic mice has been developed. The regulatory promoter element was derived from the rat cardiac MLC-2 gene. 2100 bp of the 5' regulatory MLC-2 sequences were found to drive constitutive cardiac expression of a firefly luciferase reporter gene from early tubular heart formation. During ventricular loop and septum formation luciferase activity was 10-fold upregulated in comparison to steady-state levels observed 10 days after birth. No luciferase activity was detectable in any other muscle or non-muscle tissue of transgenic mice. These data suggest that the 2.1 kb DNA sequences of the 5' flanking region of the cardiac MLC-2 gene contain sufficient regulatory elements for a selective gene expression in cardiac myocytes from embryogenesis. The transgenic model should aid in determining the influences of pathogenic gene products on developing and mature heart muscle to elucidate the etiology of myocardial diseases such as cardiomyopathies.
Animals↗
Molecular mechanisms in the pathogenesis of enteroviral heart disease: acute and persistent infections.
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Animals↗
Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice.
Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2-luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene, no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice. This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular mechanisms of myocardial diseases such as cardiomyopathies.
Animals↗
Deletion of an N-terminal regulatory domain of the c-abl tyrosine kinase activates its oncogenic potential.
The requirements for the oncogenic conversion of the c-abl proto-oncogene have been determined by the expression of N-terminal deleted forms and viral gag-fused forms of the c-abl proteins from a selectable retroviral vector. To activate the transforming potential of c-abl, it is necessary that (i) specific N-terminal amino acids are deleted to release the kinase from negative regulation in vivo; (ii) an N-terminal myristylation site is part of the activated kinase; (iii) the fatty-acylated, activated kinase is overproduced. The N-terminal amino acids found to be necessary for the cellular inhibition of c-abl tyrosine phosphorylation are part of a homologous region present in many non-receptor tyrosine kinases, the v-crk oncogene and phospholipase C-II. Overproduction of a deregulated and myristylated c-abl tyrosine kinase induces the transformation of NIH 3T3 cells.
Abelson murine leukemia virus↗