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SERCA2 and ANF Promoter-Activity Studies in Hypertrophic Cardiomyocytes Using Liposome-, Gene Gun-, and Adenovirus-Mediated Gene Transfer

  • Chapter
The Hypertrophied Heart

Part of the book series: Progress in Experimental Cardiology ((PREC,volume 3))

Summary

Myocardial hypertrophy is known as the process of enlargement of ventricular cells, which is also accompanied by changes in the phenotype. The latter changes include, e.g., downregulation of the expression of sarcoplasmatic reticulum Ca2+ ATPase (SERCA2), phospholamban (PL), and β-adrenergic receptor and upregulation of the expression of atrial natriuretic factor (ANF) and β-myosin heavy chain (ß-MHC). Analysis of the transcriptional regulation of a promoter fragment of the SERCA2 gene using liposome-mediated transfection revealed that the SERCA2 gene may not respond to the general increase in transcription upon stimulation of neonatal rat cardiomyocytes by 10-8M endothelin-1. Liposome-mediated transfection used in these promoter activity studies yields no more than 1% transfection efficiency (the percentage of cells expressing the transgene). To obtain higher efficiency, we set out to develop the gene-gun biolistics method for transfection of cardiomyocytes. An efficiency up to approximately 10% can be achieved by the gene gun as tested using a RSV-β-Gal construct. Here, we demonstrate the efficacy of the method by use of an endothelin-1 inducible ANF promoter fragment coupled to a CAT reporter. Therefore, gene-gun biolistics is ideally suited as a quick and reliable method to test DNA constructs on their activity. The ANF promoter is normally only active to a very low extent in ventricular adult cells; it is upregulated by hypertrophic stimuli. We used the latter property for generating DNA constructs encompassing the antisense PL gene under the control of the endothelin-1 inducible ANF promoter fragment. Adenovirus infection with almost 100% efficiency is required to measure the functional consequences of the overexpressed antisense PL. Here, we present the first results with the ANF-promoter-PL-antisense adenovirus infection of rat neonatal cardiomyocytes.

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Authors and Affiliations

  1. Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR, Rotterdam, The Netherlands

    Karin Eizema, Han A. A. Van Heugten, Karel Bezstarosti, Marga C. Van Setten & Jos M. J. Lamers

  2. Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Free University, Hindenburgdamm 30, D-122200, Berlin, Germany

    Sonja Schneider-Rasp & Wolfgang C. Poller

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  1. Karin Eizema
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  2. Han A. A. Van Heugten
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  3. Karel Bezstarosti
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  4. Marga C. Van Setten
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  5. Sonja Schneider-Rasp
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  6. Wolfgang C. Poller
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  7. Jos M. J. Lamers
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Editor information

Editors and Affiliations

  1. Department of Internal Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan

    Nobuakira Takeda M.D., Ph.D. (Associate Professor) (Associate Professor)

  2. Jikei University School of Medicine, Tokyo, Japan

    Makoto Nagano M.D., Ph.D. (Professor Emeritus) (Professor Emeritus)

  3. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

    Naranjan S. Dhalla Ph.D., M.D. (Hon.), D.Sc. (Hon) (Distinguished Professor and Director) (Distinguished Professor and Director)

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Eizema, K. et al. (2000). SERCA2 and ANF Promoter-Activity Studies in Hypertrophic Cardiomyocytes Using Liposome-, Gene Gun-, and Adenovirus-Mediated Gene Transfer. In: Takeda, N., Nagano, M., Dhalla, N.S. (eds) The Hypertrophied Heart. Progress in Experimental Cardiology, vol 3. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-4423-4_5

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  • DOI: https://doi.org/10.1007/978-1-4615-4423-4_5

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4613-6991-2

  • Online ISBN: 978-1-4615-4423-4

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