Modulation of cardiomyocyte electrical properties using regulated bone morphogenetic protein-2 expression.

Because cardiomyocytes lose their ability to divide after birth, any subsequent cell loss or dysfunction results in pathologic cardiac rhythm initiation or impulse conduction. Strategies to restore and control the electrophysiological activity of the heart may, therefore, greatly affect the regeneration of cardiac tissue functionality. Using lentivirus-derived particles to regulate the bone morphogenetic protein-2 (BMP-2) gene expression in a pristinamycin- or gaseous acetaldehyde-inducible manner, we demonstrated the adjustment of cardiomyocyte electrophysiological characteristics. Complementary metal oxide semiconductor-based high-density microelectrode arrays (HD-MEAs) were used to monitor the electrophysiological activity of neonatal rat cardiomyocytes (NRCs) cultured as monolayers (NRCml) or as microtissues (NRCmt). NRCmt more closely resembled heart tissue physiology than did NRCml and could be conveniently monitored using HD-MEAs because of their ability to detect low-signal events and to sub-select the region of interest, namely, areas where the microtissues were placed. Cardiomyocyte-forming microtissues, transduced using lentiviral vectors encoding BMP-2, were capable of restoring myocardial microtissue electrical activity. We also engineered NRCmt to functionally couple within a cardiomyocyte monolayer, thus showing pacemaker-like activity upon local regulation of transgenic BMP-2 expression. The controlled expression of therapeutic transgenes represents a crucial advance for clinical interventions and gene-function analysis.

[1]  I. Harary,et al.  In vitro studies on single beating rat heart cells. I. Growth and organization. , 1963, Experimental cell research.

[2]  I HARARY,et al.  In vitro studies on single beating rat heart cells. II. Intercellular communication. , 1963, Experimental cell research.

[3]  R C Barr,et al.  Extracellular Potentials Related to Intracellular Action Potentials during Impulse Conduction in Anisotropic Canine Cardiac Muscle , 1979, Circulation research.

[4]  B. Höijer,et al.  Improved methods for automatic monitoring of contracting heart cells in culture. , 1980, Journal of biochemical and biophysical methods.

[5]  D. Simpson,et al.  Assembly and remodelling of myofibrils and intercalated discs in cultured neonatal rat heart cells. , 1986, Journal of cell science.

[6]  V. Rosen,et al.  Novel regulators of bone formation: molecular clones and activities. , 1988 .

[7]  Effects of ethanol on voltage-sensitive Na-channels in cultured skeletal muscle: up-regulation as a result of chronic treatment. , 1990, The Journal of pharmacology and experimental therapeutics.

[8]  W. Giles,et al.  Alpha 1‐adrenoceptors reduce background K+ current in rabbit ventricular myocytes. , 1991, The Journal of physiology.

[9]  E. Lakatta,et al.  Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes. , 1991, Circulation research.

[10]  J. Molkentin,et al.  Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo. , 1993, The Journal of biological chemistry.

[11]  N. Goldschlager,et al.  Medical progress : cardiac pacing , 1996 .

[12]  M. Sanguinetti,et al.  Molecular Genetic Insights into Cardiovascular Disease , 1996, Science.

[13]  A. Bradley,et al.  Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. , 1996, Development.

[14]  B. Hogan,et al.  Bone morphogenetic proteins: multifunctional regulators of vertebrate development. , 1996, Genes & development.

[15]  L. Leinwand,et al.  Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research. , 1997, Circulation.

[16]  C. Hennekens.  Increasing burden of cardiovascular disease: current knowledge and future directions for research on risk factors. , 1998, Circulation.

[17]  Martin Fussenegger,et al.  Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells , 1998, Nature Biotechnology.

[18]  A. Kleber,et al.  Slow conduction in cardiac tissue, I: effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. , 1998, Circulation research.

[19]  E. Ehler,et al.  Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting. , 1999, Molecular biology of the cell.

[20]  L. Kedes,et al.  A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. , 1999, Journal of molecular and cellular cardiology.

[21]  E. Morkin.  Control of cardiac myosin heavy chain gene expression , 2000, Microscopy research and technique.

[22]  M. Fussenegger,et al.  Streptogramin-based gene regulation systems for mammalian cells , 2000, Nature Biotechnology.

[23]  J. Hell,et al.  Regulation of Cardiac L-Type Calcium Channels by Protein Kinase A and Protein Kinase C , 2000, Circulation research.

[24]  L. Leinwand,et al.  Myosin heavy chain isoform expression in the failing and nonfailing human heart. , 2000, Circulation research.

[25]  Y. Fujio,et al.  Bone Morphogenetic Protein-2 Inhibits Serum Deprivation-induced Apoptosis of Neonatal Cardiac Myocytes through Activation of the Smad1 Pathway* , 2001, The Journal of Biological Chemistry.

[26]  D. Rice,et al.  Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart , 2001, Cell and Tissue Research.

[27]  J. Psotová,et al.  Neonatal rat cardiomyocytes--a model for the study of morphological, biochemical and electrophysiological characteristics of the heart. , 2001, Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia.

[28]  J. Camm,et al.  New approaches to antiarrhythmic therapy: emerging therapeutic applications of the cell biology of cardiac arrhythmias(1). , 2001, Cardiovascular research.

[29]  M. Fussenegger,et al.  The Impact of Mammalian Gene Regulation Concepts on Functional Genomic Research, Metabolic Engineering, and Advanced Gene Therapies , 2001, Biotechnology progress.

[30]  J. Bailey,et al.  Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes , 2001, Basic Research in Cardiology.

[31]  H. Bujard,et al.  Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. , 2002, Molecular therapy : the journal of the American Society of Gene Therapy.

[32]  M. Fussenegger,et al.  Novel promoter/transactivator configurations for macrolide‐ and streptogramin‐responsive transgene expression in mammalian cells , 2002, The journal of gene medicine.

[33]  A. Lever,et al.  Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo , 2002, Basic Research in Cardiology.

[34]  I. Komuro,et al.  A role for bone morphogenetic protein signaling in cardiomyocyte differentiation. , 2002, Trends in cardiovascular medicine.

[35]  C. Kahn,et al.  Regulation of Myocardial Contractility and Cell Size by Distinct PI3K-PTEN Signaling Pathways , 2002, Cell.

[36]  Peter Wernet,et al.  Repair of Infarcted Myocardium by Autologous Intracoronary Mononuclear Bone Marrow Cell Transplantation in Humans , 2002 .

[37]  T. Johansen,et al.  Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS , 2002, Gene Therapy.

[38]  M. Fussenegger,et al.  Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. , 2002, Nucleic acids research.

[39]  Ulrich Egert,et al.  Biological application of microelectrode arrays in drug discovery and basic research , 2003, Analytical and bioanalytical chemistry.

[40]  J. Hancox,et al.  Inhibition of HERG K+ Current and Prolongation of the Guinea‐Pig Ventricular Action Potential by 4‐Aminopyridine , 2003, The Journal of physiology.

[41]  B. Chandrasekar,et al.  BMP‐2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase‐dependent manner , 2003, FEBS letters.

[42]  A. Abbott.  Cell culture: Biology's new dimension , 2003, Nature.

[43]  M. Rubart,et al.  Physiological Coupling of Donor and Host Cardiomyocytes After Cellular Transplantation , 2003, Circulation research.

[44]  M. Rubart,et al.  Myocyte and myogenic stem cell transplantation in the heart. , 2003, Cardiovascular research.

[45]  H. Bujard,et al.  Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo , 2003, Gene Therapy.

[46]  Martin Fussenegger,et al.  Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. , 2003, Biotechnology and bioengineering.

[47]  Ulrich Egert,et al.  Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology Estimation of Action Potential Changes from Field Potential Recordings in Multicellular Mouse Cardiac Myocyte Cultures Key Words Microelectrode Array @bullet Action Potential Upstroke @bullet Action P , 2022 .

[48]  L. Mahimainathan,et al.  Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2 (BMP-2)-induced Myocyte Enhancer Factor 2A-dependent Transcription of BMP-2 Gene in Cardiomyocyte Precursor Cells* , 2003, Journal of Biological Chemistry.

[49]  N. Déglon,et al.  Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum. , 2003, Human molecular genetics.

[50]  A. Leaf,et al.  Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels. , 2004, European journal of pharmacology.

[51]  James P. Freyer,et al.  The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model , 2004, Journal of biomolecular screening.

[52]  M. Fussenegger,et al.  Approaches for trigger-inducible viral transgene regulation in gene-based tissue engineering. , 2004, Current opinion in biotechnology.

[53]  Heribert Bohlen,et al.  Determination of electrical properties of ES cell-derived cardiomyocytes using MEAs. , 2004, Journal of electrocardiology.

[54]  Martin Fussenegger,et al.  Design of artificial myocardial microtissues. , 2004, Tissue engineering.

[55]  Martin Fussenegger,et al.  Gas-inducible transgene expression in mammalian cells and mice , 2004, Nature Biotechnology.

[56]  Shuguang Zhang,et al.  Beyond the Petri dish , 2004, Nature Biotechnology.

[57]  J. Mallet,et al.  A single lentivirus vector mediates doxycycline-regulated expression of transgenes in the brain. , 2004, Human gene therapy.

[58]  Heping Cheng,et al.  Subtype-specific β-adrenoceptor signaling pathways in the heart and their potential clinical implications , 2004 .

[59]  R. Quian Quiroga,et al.  Unsupervised Spike Detection and Sorting with Wavelets and Superparamagnetic Clustering , 2004, Neural Computation.

[60]  M. Fussenegger,et al.  Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression. , 2004, Nucleic acids research.

[61]  Y. Rudy,et al.  Basic mechanisms of cardiac impulse propagation and associated arrhythmias. , 2004, Physiological reviews.

[62]  M. Fussenegger,et al.  Dual‐regulated myoD‐ and msx1‐based interventions in C2C12‐derived cells enable precise myogenic/osteogenic/adipogenic lineage control , 2004, The journal of gene medicine.

[63]  Michael R Rosen,et al.  Genes, stem cells and biological pacemakers. , 2004, Cardiovascular research.

[64]  Rona Shofti,et al.  Electromechanical integration of cardiomyocytes derived from human embryonic stem cells , 2004, Nature Biotechnology.

[65]  M. Fussenegger,et al.  REVIEW: Synergies of microtissue design, viral transduction and adjustable transgene expression for regenerative medicine , 2004, Biotechnology and applied biochemistry.

[66]  M. Fussenegger,et al.  Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts. , 2004, Nucleic acids research.

[67]  Martin Fussenegger,et al.  In vivo transduction of HIV‐1‐derived lentiviral particles engineered for macrolide‐adjustable transgene expression , 2005, The journal of gene medicine.

[68]  D. Allen,et al.  Myocyte Enhancer Factor-2 and Serum Response Factor Binding Elements Regulate Fast Myosin Heavy Chain Transcription in Vivo* , 2005, Journal of Biological Chemistry.

[69]  M. Fussenegger,et al.  Autoregulated, bidirectional and multicistronic gas-inducible mammalian as well as lentiviral expression vectors. , 2005, Journal of biotechnology.

[70]  I. Kawase,et al.  Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury , 2005, Circulation.

[71]  A. Kleber,et al.  The shape of the electrical action-potential upstroke: a new aspect from optical measurements on the surface of the heart. , 2005, Circulation research.

[72]  M. Fussenegger,et al.  Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles. , 2005, Metabolic engineering.

[73]  S. Priori,et al.  Role of Genetic Analyses in Cardiology: Part I: Mendelian Diseases: Cardiac Channelopathies , 2006, Circulation.

[74]  M. Rubart,et al.  Cardiac regeneration: repopulating the heart. , 2006, Annual review of physiology.

[75]  M. Fussenegger,et al.  Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues , 2006, Biotechnology and bioengineering.

[76]  E. Lakatta,et al.  Subtype-specific α1- and β-adrenoceptor signaling in the heart , 2006 .

[77]  Martin Fussenegger,et al.  Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats. , 2006, Tissue engineering.

[78]  Michael R Rosen,et al.  I(f) and the biological pacemaker. , 2006, Pharmacological research.

[79]  A. Moorman,et al.  Role of bone morphogenetic proteins in cardiac differentiation. , 2007, Cardiovascular research.

[80]  Dong Liu,et al.  Bone morphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility. , 2007, Cardiovascular research.

[81]  M. Fussenegger,et al.  Novel gene switches. , 2007, Handbook of experimental pharmacology.

[82]  U. Frey,et al.  Single-chip microelectronic system to interface with living cells. , 2007, Biosensors & bioelectronics.