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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Schwartz K, Carrier L, Mercadier J-J, Lompre A-M, Boheler KR. 1993. Molecular phenotype of the hypertrophied and failing myocardium. Circulation 87:VII5–VII10.
Van Heugten HAA, De Jonge HW, Goedbloed MA, Bezstarosti K, Sharma HS, Verdouw PD, Lamers JMJ. 1995. Intracellular signaling and genetic reprogramming during development of hypertrophy in cultured cardiomyocytes. In Heart Hypertrophy and Failure Ed. NS Dhalla, GN Pierce, VN Panagia, and L Beamish, 79–92. Boston: Kluwer Academic Publishers.
Van Heugten HAA, Lamers JMJ. 1997. Changes in cardiac phenotype in hypertrophy and failure: from receptor to gene. Heart Failure Rev 2:95–106.
Komuro I.Yazaki Y. 1993. Control of cardiac gene expression by mechanical stress. Annu Rev Physiol 55:55–75.
Love MP, McMurray JJV. 1996. Endothelin in chronic heart failure: current position and future prospects. Cardiovasc Res 31:665–774.
Baker KM, Booz GW, Dostal DE. 1992. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu Rev Physiol 54:227–241.
Simpson P. 1983. Norepinephrine-stimulated hypertrophy of cultured neonatal rat cardiomyocytes. J Clin Invest 72:732–738.
Swynghedauw B. 1986. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev 66:710–771.
Morgan JP, Erny RE, Allen PD, Grossman W, Gwathmey JK. 1990. Abnormal intracellular Ca2+ handling. A major cause of systolic and diastolic dysfuntion in ventricular myocardium from patients with heart failure. Circulation 81(Suppl III):21–32.
Arai M, Matsui H, Periasamy M. 1994. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 74:555–564.
Hasenfuss G, Meyer M, Schillinger W, Preuss M, Priske B, Just H. 1997. Calcium handling proteins in the failing human heart. Basic Res Cardiol 92(Suppl 1):87–93.
Flesch M, Schwinger RHG, Schnabel P, Schiffer F., Van Gelder I, Bavendiek U, Südkamp M, Kuhn-Regnier F, Böhm M. 1996. Sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNA and protein levels in end-stage heart failure due to ischemic or dilated cardiomyopathy. J Mol Med 74:321–332.
Zarain-Herzberg A, Afzal N, Elimban V, Dhalla NS. 1996. Decreased expression of sarcoplasmic reticulum Ca2+ pump ATPase in congestive heart failure due to myocardial infarction. Mol Cell Biochem 163/164:285–290.
Linck B, Boknik P, Eschenhagen T, Müller FU, Neumann J, Nose M, Jones LR, Schmitz W, Scholz H. 1996. Messenger RNA expression and immunological quantification of phospholamban and SR-Ca2+-ATPase in failing and nonfailing human hearts. Cardiovasc Res 31:625–632.
Kovacic-Milivojevic B., Wong VSH, Gardner DG. 1996. Selective regulation of the atrial natriuretic peptide gene by individual components of the activatorprotein-1 complex. Endocrinology 137:1108–1117.
Karns LR, Kariya K, Simpson PC. 1995. M-CAT, CarG, and Sp1 elements are required for α-adrenergic induction of the skeletal α-actin promoter during cardiac myocyte hypertrophy. J Biol Chem 270:410–417.
Sadoshima J, Izumo S. 1993. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Circ Res 73:424–438.
Kariya K, Karns LR, Simpson PC. 1994. An enhancer core element mediates stimulation of the rat α-myosin heavy chain promoter by an α1-adrenergic agonist and activated protein kinase C in hypertrophy of cardiac myocytes. J Biol Chem 269:3775–3782.
Molkentin JD, Lu J-R, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. 1998. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228.
Van Heugten HAA, Bezstarosti K, Dekkers DHW, Lamen JMJ. 1994. Homologous desensitization of the endothelin-1 receptor evoked phosphoinositide response in cultured neonatal rat cardio-myocytes. J Mol Cell Cardiol 25:41–52.
Van Heugten HAA, De Jonge HW, Bezstarosti K, Sharma HS, Verdouw PD, Lamen JMJ. 1995. Intracellular signalling and genetic reprogramming during agonist-induced hypertrophy of cardiomyocytes. Ann NY Acad Sci 752:343–352.
Van Heugten HAA, Van Setten MC, Eizema K, Verdouw P, Lamers JMJ. 1998. Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes. Cardiovasc Res 37:503–514.
Antin PB, Mar JH, Ordahl CP. 1988. Single cell analysis of transfected gene expression in primary heart cell cultures containing multiple cell types,. Biotechnology 6:630–648.
Kohout TA, O’Brian JJ, Gaa ST, Lederer WJ, Rogers TB. 1996. Novel adenovirus component system that transfects cultured cardiac cells with high efficiency. Circ Res 78:971–977.
Hajjar RJ, Schmidt U, Kang JX, Matsui T, Rosenzweig A. 1997. Adenovirus gene transfer of phospholamban in isolated rat cardiomyocytes. Rescue effects by concomitant gene transfer of sarcoplasmic reticulum Ca2+ATPase. Circ Res 81:145–153.
Meyer M, Dillmann WH. 1998. Sarcoplasmic Ca2+ATPase overexpressing by adenovirus mediated gene transfer and in transgenic mice. Cardiovasc Res 37:360–366.
Hajjar RJ, Kang JX, Gwathmey JK, Rosenzweig A. 1997. Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum Ca2+ATPase in isolated rat myocytes. Circulation 95:423–429.
Yang N-S, Sun WH, McCabe D. 1996. Developing particle-mediated gene-transfer technology for research into gene therapy of cancer. Mol Med Today (Nov):476–481.
Yoshida Y, Kobayashi E, Endo H, Hamamoto T, Yamanaka T, Fujimura A, Kagawa Y 1997. Introduction of DNA into rat liver with a hand-held Gene gun: Distribution of the expressed enzyme, [32P] DNA, and Ca2+ flux. Biochem Biophys Res Commun 234:695–700.
Sanford JC. 1988. The biolistic process. Trends Biotechnol 6:299–302.
Eizema K, van Heugten HAA, Bezstarosti K, van Setten MC, Lamers JMJ. 1999. In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by Gene-Gun biolistics. Ann NY Acad Sci 874:111–124.
Eizema K, van Heugten HAA, Bezstarosti K, van Setten MC, Lamers JMJ. 2000. Endothelin-1 responsiveness of a 1.4kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun. J Mol Cell Cardiol 32:311–321.
Decock JB, Gillespie-Brown J, Parker PJ, Sugden PH, Fuller SJ. 1994. Classical, novel and atypical isoforms of PKC stimulate ANF and TRE/AP-1 regulated promoter activity in ventricular cardiomyocytes. FEBS Lett 356:275–278.
Lompre A-M, De La Bastie D, Boheler KR, Schwartz K. 1989. Characterization and expression of the rat sarcoplasmic reticulum Ca2+ATPase mRNA. FEBS Lett 249:35–41.
Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Heller Brown J, Chien KR. 1990. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for cell hypertrophy. J Biol Chem 265:20555–20562.
Kovacic-Milivojevic B, Gardner DG. 1993. Regulation of the human atrial natriuretic peptide gene in atrial cardiocytes by the transcription factor AP-1. Am J Hypertens 6:258–263.
Kovacic-Milivojevic B, Gardner DG. 1995. Fra-1, a fos gene family member that activates atrial natriuretic peptide gene transcription. Hypertension 25:679–682.
Kass-Eisler A, Falck Pedersen E, Alvira M, Revera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA. 1993. Quantitative determination of adenovirus mediated gene-delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA 90:11498–11502.
Kirshenbaum LA, Maclellan WR, Mazur W, French BA, Schneider MD. 1993. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J Clin Invest 92:381–387.
Kirshenbaum LA. 1997. Adenovirus mediated-gene transfer into cardiomyocytes. Mol Cell Biochem 172:13–21.
Hasenfuss G, Just H. 1994. Myocardial phenotype changes in heart failure: cellular and subcellular changes and their functional significance. Br Heart J 72:510–517.
Chien KR, Ibu H, Knowlton KR, Miller-Honce W, van Bilsen M, Obrein TX, Evans SM. 1993. Transcriptional regulation during cardiac growth and development. Annu Rev Physiol 55:77–95.
Qi M, Bassani JW, Bers DM, Samarel AM. 1996. Phorbol 12-myristate 13-acetate alters SR Ca(2+)-ATPase gene expression in cultured neonatal rat heart cells. Am J Physiol 271(3, Pt 2):H1031–1039.
Hartong R, Villarreal FJ, Giordano F, Hilal-Dandan R, McDonough PM, Dillmann WH. 1996. Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake. J Mol Cell Cardiol 28:2467–2477.
Eskildsen, YEG, Bezstarosti K, Dekkers DHW, Van Heugten HAA, Lamers JMJ. 1997. Cross-talk between receptor-mediated phospholipase C-β and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes. J Mol Cell Cardiol 29:2545–2559.
Clerk A, Bogoyevitch MA, Anersson MB, Sugden P. 1994. Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrin and subsequent stimulation of p42 and p44 mitogenactivated protein kinases in ventricular myocytes cultured from neonatal rat heart. J Biol Chem 269:32848–32857.
Puceat M, Hilal-Dandan R, Stulovia B, Brunton LL, Heller Brown J. 1994. Differential regulation of protein kinase C isoforms in isolated neonatal and adult cardiomyocytes. J Biol Chem 269:16938–16944.
Cadre BM, Qi M, Eble DM, Shannon TR, Bers DM, Samarel AM. 1998. Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes J Mol Cell Cardiol 30:2247–2259.
Argentin S, Ardiati A, Tremblay S, Lihrmann I, Robitaille L, Drouin J, Nemer M. 1994. Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells. Mol Cell Biol 14:777–790.
Knowlton KU, Baraccini E, Ross RR, Harris AN, Henderson SA, Evans SM, Glembotski CC, Chien KR. 1991. Coregulation of the atrial natriuretic factor and cardiac myosin-light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. J Biol Chem 266:7759 mitogen-activated 7768.
Rockman HA, Ross R, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross J, Chien KR. 1991. Segregation of atrial-specific and inducible expression of natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci USA 88:8277–8281.
Koss KL, Kranias EG. 1996. Phospholamban: a prominent regulator of myocardial contractility. Circ Res 79:1059–1063.
Simmerman HKB, Jones LR. 1998. Phospholamban: protein structure, mechanism of action and role in cardiac function. Physiol Rev 78:921–947.
Luo W, Wolska BM, Grupp H, Harrier JM, Haghighi K, Ferguson DG, Slack JP, Grupp G, Doetschman T, Solaro RJ, Kranias EG. 1996. Phospholamban gene dosage effects in the mammalian heart. Circ Res 78:839–847.
Wu J, Kovacic-Milivojevic B, La Pointe MC, Nakamura K, Gardner DB. 1991. Cisspreactive determinants of cardiacspecific expression in the human atrial natriuretic peptide gene. Mol Endocrinol 5:1311–1322.
Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee K-H, Gwathmey JK, Dec GW, Semigran MJ, Rosenzweig A. 1998. Modulation of ventricular function through gene transfer in vivo. Proc Nad Acad Sci USA 95:5251–5256.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Springer Science+Business Media New York
About this chapter
Cite this chapter
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
Download citation
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
eBook Packages: Springer Book Archive