Michael S. Parmacek

Expression of recombinant genes in myocardium in vivo after direct injection of DNA.

The ability to program recombinant gene expression in cardiac myocytes in vivo holds promise for the treatment of many inherited and acquired cardiovascular diseases. In this report, we demonstrate that a recombinant beta-galactosidase gene under the control of the Rous sarcoma virus promoter can be introduced into and expressed in adult rat cardiac myocytes in vivo by the injection of purified plasmid DNA directly into the left ventricular wall. Cardiac myocytes expressing recombinant beta-galactosidase were detected histochemically in rat hearts for at least 4 weeks after injection of the beta-galactosidase gene. These results demonstrate the potential of this method of somatic gene therapy for the treatment of cardiovascular disease.

GATA-4 Activates Transcription Via Two Novel Domains That Are Conserved within the GATA-4/5/6 Subfamily

GATA-4 is one of the earliest developmental markers of the precardiac mesoderm, heart, and gut and has been shown to activate regulatory elements controlling transcription of genes encoding cardiac-specific proteins. To elucidate the molecular mechanisms underlying the transcriptional activity of the GATA-4 protein, structure-function analyses were performed. These analyses revealed that the C-terminal zinc finger and adjacent basic domain of GATA-4 is bifunctional, modulating both DNA-binding and nuclear localization activities. The N terminus of the protein encodes two independent transcriptional Activation Domains (amino acids 1-74 and amino acids 130-177). Amino acid residues were identified within each domain that are required for transcriptional activation. Finally, we have shown that regions of Xenopus GATA-5 and −6 corresponding to Activation Domains I and II, respectively, function as potent transcriptional activators. The identification and functional characterization of two evolutionarily conserved transcriptional Activation Domains within the GATA-4/5/6 subfamily suggests that each of these domains modulates critical functions in the transcriptional regulatory program(s) encoded by GATA-4, −5, and −6 during vertebrate development. As such these data provide novel insights into the molecular mechanisms that control development of the heart.

Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor.

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

17 – GATA Transcription Factors and Cardiac Development

Publisher Summary This chapter summarizes the current understanding of the role of three GATA family transcription factors, GATA-4,-5, and -6, in cardiovascular development. It is found that GATA family members expressed in temporally distinct patterns in the developing mammalian heart. In addition, GATA-6 is expressed in both arterial and venous SMCs, suggesting a potential role for this protein in the development of the vasculature. GATA-4,-5, and -6 can each bind to the transcriptional regulatory regions of multiple cardiac promoters and enhancers, and transactivate these transcriptional regulatory elements in nonmuscle cells. Each of these factors share two unique and independent transcriptional activation domains. Gene targeting experiments have demonstrated a necessary role for GATA-4 in regulating the second stage of cardiac development in the migration of specified procardiomyocytes from the dorsolateral region of the embryo to form the ventral linear heart tube. Chimera experiments suggest that GATA-4 is required to initiate and/or maintain the ventral morphogenic signal rather than to act as a regulator of cardiomyocyte responsiveness to this signal. GATA-6 is also required for early embryonic viability and its precise role in cardiovascular development is currently under investigation. In contrast, GATA-5 does not appear to be required for the development of the heart and vasculature. Finally, preliminary evidence suggests that GATA-4 and GATA-6 may belong to a common developmental pathway in which GATA-4 normally down regulates the expression of GATA-6 in the heart and extra embryonic membranes.

Direct gene transfer into cardiac myocytes in vivo

Abstract Recent studies have demonstrated that cardiac and skeletal myocytes share the ability to take up and stably express plasmid DNA injected directly into myocardium or skeletal muscle in vivo . Although this is a relatively inefficient process, with less than 1% of the myocytes expressing the injected recombinant DNA, expression in these cells is stable for periods of at least 6 months. The majority of the injected DNA is maintained in myocytes as an episome and apparently does not undergo DNA replication. The direct DNA injection approach has been used to map cardiac-specific transcriptional regulatory elements in cellular promoter/enhancers. Expression of recombinant proteins in the heart following direct DNA injection also holds promise for the treatment of a variety of acquired and inherited cardiovascular diseases.