Current Search:  Cardiomyocytes (x)

View All Items

  • CSV Spreadsheet
(1 - 3 of 3)
ZINC-FINGER PROTEIN MCPIP IN CELL DEATH AND DIFFERENTIATION
Title
ZINC-FINGER PROTEIN MCPIP IN CELL DEATH AND DIFFERENTIATION.
Creator
Younce, Craig, Kolattukudy, Pappachan, University of Central Florida
Abstract / Description
Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted...
Show moreMonocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure. A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ. Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM's resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4'PBA prevented high glucose-induced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3'methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.
Show less
Date Issued
2009
Identifier
CFE0002888, ucf:48027
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002888
Cell Printing: An Effective Advancement for the Creation of um Size Patterns for Integration into Microfluidic BioMEMs Devices
Title
Cell Printing: An Effective Advancement for the Creation of um Size Patterns for Integration into Microfluidic BioMEMs Devices.
Creator
Aubin, Megan, Hickman, James, Coffey, Kevin, Lambert, Stephen, University of Central Florida
Abstract / Description
The Body-on-a-Chip (BoaC) is a microfluidic BioMEMs project that aims to replicate major organs of the human body on a chip, providing an in vitro drug testing platform without the need to resort to animal model testing. Using a human model also provides significantly more accurate drug response data, and may even open the door to personalized drug treatments. Microelectrode arrays integrated with human neuronal or human cardiac cells that are positioned on the electrodes are essential...
Show moreThe Body-on-a-Chip (BoaC) is a microfluidic BioMEMs project that aims to replicate major organs of the human body on a chip, providing an in vitro drug testing platform without the need to resort to animal model testing. Using a human model also provides significantly more accurate drug response data, and may even open the door to personalized drug treatments. Microelectrode arrays integrated with human neuronal or human cardiac cells that are positioned on the electrodes are essential components for BoaC systems. Fabricating these substrates relies heavily on chemically patterned surfaces to control the orientation and growth of the cells. Currently, cells are plated by hand onto the surface of the chemically patterned microelectrode arrays. The cells that land on the cytophobic 2-[Methoxy(Polyethyleneoxy)6-9Propyl]trimethoxysilane (PEG) coating die and detach from the surface, while the cells that land on the cytophilic diethylenetriamine (DETA) coating survive and attach to the surface exhibiting normal physiology and function. The current technique wastes a significant amount of cells, some of which are extremely expensive, and is labor intensive. Cell printing, the process of dispensing cells through a 3D printer, makes it possible to pinpoint the placement of cells onto the microelectrodes, drastically reducing the number of cells utilized. Scaled-up manufacturing is also possible due to the automation capabilities provided by printing. In this work, the specific conditions for printing each cell type is unique, the printing of human motoneurons, human sensory neurons and human cardiac cells was investigated. The viability and functionality of the printed cells are demonstrated by phase images, immunostaining and electrical signal recordings. The superior resolution of cell printing was then taken one step further by successfully printing two different cell types in close proximity to encourage controlled innervation and communication.
Show less
Date Issued
2017
Identifier
CFE0007390, ucf:52074
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007390
PATTERNED CELL CULTURES FOR HIGH THROUGHPUT STUDIES OF CELL ELECTROPHYSIOLOGY AND DRUG SCREENING APPLICATIONS
Title
PATTERNED CELL CULTURES FOR HIGH THROUGHPUT STUDIES OF CELL ELECTROPHYSIOLOGY AND DRUG SCREENING APPLICATIONS.
Creator
Natarajan, Anupama, Hickman, James, University of Central Florida
Abstract / Description
Over the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug...
Show moreOver the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug screening assays in the pharmacology industry and studies of cell-surface interactions are complicated and invasive. Most cell physiology studies are performed using conventional patch-clamp techniques or random networks cultured on silicon devices such as Microelectrode Arrays (MEAs) and Field Effect transistors (FETs). The objective of this study was to develop high-throughput in vitro platforms that could be used to analyze cell function and their response to various stimuli. Our hypothesis was that by utilizing surface modification to provide external guidance cues for various cell types and by controlling the cell environment in terms of culture conditions, we could develop an in vitro hybrid platform for sensing and testing applications. Such a system would not only give information regarding the surface effects on the growth and behavior of cells for implant development applications, but also allow for the study of vital cell physiology parameters like conduction velocity in cardiomyocytes and synaptic plasticity in neuronal networks. This study outlines the development of these in vitro high throughput systems that have varied applications ranging from tissue engineering to drug development. We have developed a simple and relatively high-throughput method in order to test the physiological effects of varying chemical environments on rat embryonic cardiac myocytes in order to model the degradation effects of polymer scaffolds. Our results, using our simple test system, are in agreement with earlier observations that utilized a complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. Traditional multielectrode extracellular recording methods were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; for example, to enable the measurement of conduction velocity, refractory period after action potentials or to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, the patterning of cardiac myocyte monolayers increased the information content of traditional multielectrode measurements. Patterning techniques with self-assembled monolayers on microelectrode arrays were also used to study the physiological properties of hippocampal networks with functional uni-directional connectivity, developed to study the mono-synaptic connections found in the dentate gyrus. Results indicate that changes in synaptic connectivity and strength were chemically induced in these patterned hippocampal networks. This method is currently being used for studying long term potentiation at the cellular level. For this purpose, two cell patterns were optimized for cell migration onto the pattern as demonstrated by time lapse studies, and for supporting the best pattern formation and cell survival on these networks. The networks formed mature interconnected spiking neurons. In conclusion, this study demonstrates the development and testing of in vitro high-throughput systems that have applications in drug development, understanding disease models and tissue engineering. It can be further developed for use with human cells to have a more predictive value than existing complex, expensive and time consuming methods.
Show less
Date Issued
2010
Identifier
CFE0003384, ucf:48437
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003384