Faculty Bibliography

Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables

The mechanisms regulating cardiac muscle differentiation and development are incompletely understood. To examine the relationships between cardiocyte proliferation and differentiation, we tested the ability of a fragment from the rat beta myosin heavy-chain (MHC beta) gene to correctly target expression of a thermolabile simian virus 40 large tumor antigen allele (tsA58) in the developing mouse. Transgene expression in the heart was observed as early as 10 days postconception and was developmentally regulated in parallel with the endogenous MHC beta gene. Expression was also detected in developing skeletal muscle, although at low levels. Despite the temperature sensitivity of the mutant large tumor antigen protein, a subset of transgenic mice in several lineages developed marked cardiac and skeletal myopathies

Gap junctions are specialized regions of adjoining cell membranes composed of numerous intercellular low-resistance channels. In the heart, these channels electrotonically couple adjacent myocytes and synchronize the cardiac action potential. Signaling through gap junction channels may also influence embryogenesis and development. Recent studies have identified the connexin gene family whose protein products assemble to form gap junction channels. Studies of connexin gene expression and function are providing new insights into the behavior of gap junction channels in the heart.

The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with unitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels

Connexins are protein subunits that constitute gap junction channels. Two members of this gene family, connexin43 (Cx43) and connexin32 (Cx32), are abundantly expressed in the heart and liver, respectively. Human genomic DNA analysis revealed the presence of two loci for Cx43: an expressed gene and a processed pseudogene. The expressed gene (GJA1) was mapped to human chromosome 6 and the pseudogene (GJA1P) to chromosome 5. To determine whether Cx32 was linked to Cx43, somatic cell hybrids were analyzed by polymerase chain reaction and hybridization, resulting in the assignment of the gene for Cx32 (GJB1) to the X chromosome at Xp11----q22. Comparison of the structures of connexin genes suggests that members of this multigene family arose from a single precursor, but evolved to distinct chromosomal locations

Connexin43 is the predominant gap junction protein expressed in the heart. To determine the relation between cardiac maturation and gap junction gene expression, the developmental profiles of connexin43 mRNA and protein were examined in the rat heart. Connexin43 mRNA levels accumulate progressively (eightfold) during embryonic and early neonatal stages, accompanied by a parallel, but temporally delayed, accumulation of connexin43 protein (15-fold). As the heart matures further, both mRNA and protein levels subsequently decline, to about 50% and 30% of their maximum levels, respectively. These observations suggest that increases in intercellular coupling that characterize cardiac development do not depend solely on modulation of connexin43 gene expression, but rather are likely to involve organization of gap junction channels into the intercalated disc

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes