Bioengineering Core for Cellular and Tissue Models

细胞和组织模型的生物工程核心

基本信息

批准号：
7905102
负责人：
Andrew D. McCulloch
金额：
$ 31.07万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7905102
关键词：
Acetates Actinin Action Potentials Adult Air Albumins Algorithms Anisotropy Antibodies Aorta Area Arrhythmia Atomic Force Microscopy Back Binding Biological Assay Biological Preservation Biomedical Engineering Biotechnology Bite Bolus Infusion Bovine Serum Albumin Buffers Calcium Caliber Cannulas Carbon Dioxide Cardiac Cardiac Myocytes Cardiac Output Cardiomyopathies Cardioplegic Solutions Catheters Cathodes Cell Adhesion Cell Culture Techniques Cell Line Cell Shape Cell membrane Cell-Matrix Junction Cells Cellular Morphology Cervical Chemicals Chest Chronic Code Collagen Collagen Type I Collection Complementary DNA Computer software Confocal Microscopy Contracts Coronary Coupled Creatine Culture Media Custom Cyclic AMP-Dependent Protein Kinases Cytolysis Cytoskeleton DNA Data Deoxyribonucleases Deposition Devices Diacetyl Diagnostic Imaging Diffusion Dimensions Dissection Dissociation Distal Dry Ice Dyes Edetic Acid Electric Capacitance Electrical Resistance Electrodes Electronics Elements Engineering Environment Enzymes Epicardium Epitopes Equation Equilibrium Event Female Fibroblasts Fibronectins Figs - dietary Fluorescein Fluoresceins Fluorescence Fluorescence Resonance Energy Transfer Force of Gravity Formaldehyde Freezing Frequencies Fura-2 Gases Gel Gene Expression Gene Transfer Genomics Glucose Glutamine Glutaral Goat Growth Heart Heart Atrium Heating Heparin Histology Hour Housekeeping Gene Hybridomas Hydrophobic Surfaces Hypertrophy Hypoxia Ice Image Image Analysis Immersion Investigative Technique Immunoblotting Immunoglobulin G Immunoglobulin M Incidence Incubators Infusion Pumps Injection of therapeutic agent Insulin Interphase Cell Intraperitoneal Injections Ionophores Isoflurane Isometric Exercise Isothiocyanates Joint Dislocation Kinetics Krebs-Henseleit solution Label Laboratories Laminin Lateral Least-Squares Analysis Left Left ventricular structure Length Levocarnitine Light Lighting Liquid substance Location Lysine Magnesium Maps Masks Measurable Measurement Measures Mechanics Mediating Membrane Membrane Potentials Messenger RNA Metals Methods MicroRNAs Microfabrication Microfluidics Microscope Microscopy Minor Mitral Valve Modeling Modification Molds Molecular Molecular Probes Monitor Morphologic artifacts Morphology Motion Mus Muscle Muscle Cells Muscle Fibers Muscle Rigidity Myocardial Myocardium N-terminal Nature Needles Neonatal Noise Nonparametric Statistics Normal Cell Nucleic Acids Nutrient Optics Oryctolagus cuniculus Output Oxygen Pathway interactions Pattern Penicillins Perfusion Phase Phenotype Phosphate Buffer Phosphorylation Phosphothreonine Phosphotransferases Photometry Physiologic pulse Physiological Plastics Platinum Polyethylene Glycols Polymers Polystyrenes Positioning Attribute Preparation Pressure Transducers Printing Procedures Prolate Protein Analysis Proteins Protocols documentation RNA RNA purification Rattus Reaction Reagent Recombinant Proteins Recombinants Refit Refractory Relative (related person)Reporter Reporting Resistance Resolution Response to stimulus physiology Rest Reverse Transcription Ribonucleases Right atrial structure Right ventricular structure Saline Sampling Series Serum Serum Proteins Signal Transduction Silastic Silicon Silicone Elastomers Silicones Site Solutions Specific qualifier value Specimen Speed Staging Staining method Stainless Steel Stains Steam Stimulus Stress Stretching Structure Succinimides Surface Surgical sutures Suspension substance Suspensions Syringes System Systole Taurine Techniques Teflon Temperature Testing Thick Time Tissue Extracts Tissue Model Tissue Sample Tissues Titanium Torque Traction Training Transducers Transfection Transgenic Organisms Triton X100 Tube Ultraviolet Rays United States National Institutes of Health Ventricular Ventricular Tachycardia Videotape Viral Width Work absorption antibody conjugate attenuation base calcium indicator cell fixing cell growth cell preparation cellular imaging charge coupled device camera cold temperature connectin cryostat data acquisition diacetylmonoxime digital digital video recording egg flexibility fluorescence imaging image registration improved in vitro Model insight instrument interest lens male method development micromanipulator mutant novel optical imaging papillary muscle platinum electrode polyacrylamide polyacrylamide gels polydimethylsiloxane potassium cardioplegic solution preconditioning premature pressure prevent programs protein expression ratiometric research study response retinal rods seal software development time use tissue culture titanium dioxide tool two-dimensional vector voltage

项目摘要

the time to compute a full displacement map was reduced to less than 10 seconds for a 256x256 pixel image. The most likely traction force that can cause this displacement was found by generating a matrix of the Boussinesq equations in two dimensions, which describe the displacements at every point in a purely elastic half space that are produced by a force shear to the surface acting over a defined area (Butler et al., 2002; Dembo and Wang, 1999; Marganski et al., 2003). By limiting traction nodes to locations inside of the resting cell outline, an over-specified system was generated in which more displacement vectors were known than traction locations. By using a method of Tikhonov regularization, which balances the least squares error of a displacement fit to traction magnitudes and directions with a force balance, torque balance and penalty for high magnitudes, a traction map is generated that minimizes the effects of noise and non-uniformity of the displacement map due to non-uniformity in the random distribution of markers in the gel. C3.1.f. Collagen Gel Preparation. Collagen and neutralizing solution (100 mM Hepes, pH 7.3 in 2* PBS) are kept at 4¿C until needed. Adult cardiac myocytes are isolated and allowed to aggregate by gravity at the bottom of a 15ml tube. Cells are diluted to 1 million 100,000 per ml. Collagen gels have a final concentration of 2 mg/ml. Media, neutralizing solution and then collagen are added to the tube containing the cells working on ice. The final solution quickly becomes viscous and takes on a yellow/pinkish hue. We pipette 8 ml per stretching or traction force plate and place the plate in the incubator, at 37¿C. The collagen polymerizes around the cells in 30-60 minutes, and 5 ml media is then carefully added on top of the gel. Media is changed every 3- 214 Knowlton, Kirk U. 4 days. For immunoblotting of adult cardiac myocytes cultured in 3D collagen gels, cells are detached with 0.5 mM EDTA in calcium and magnesium-free PBS and media is removed. Next 1 ml 2* lysis buffer is added to the gel, the cells are lysed and the lysate centrifuged at 4¿C for 12 minutes. 30 ul 3* Laemmli sample buffer is added to 60 ul supernatant, heated for 3 minutes at 100¿C, and loaded into an SDS-PAGE gel for protein analysis. NEW METHOD DEVELOPMENT: We will extend these traction force microscopy methods (demonstrated in section C2.1.c) from neonatal rat ventricular myocytes to apply to them neonatal and adult mouse ventricular myocytes. Having previously successfully plated and stretched mouse neonatal ventricular myocytes (Knoll et al., 2002), we expect these traction force methods to apply to mouse neonatal myocytes without significant modification. However, for adult myocytes, we will also need to add a layer of collagen gel containing the adult myocytes on top of the polyacrylamide substrate to achieve adequate cell-matrix adhesion. Given that we have seen isolated adult myocytes can be adhered at their ends with poly-lysine and generate physiological systolic tension without detaching (Bluhm et al., 1995), and given that we have also seen that neonatal myocytes can generate physiological active stresses when adhered polyacrylamide gels (section C2.1.C), we are confident that conditions can be created that will allow adult murine ventricular myocytes to be sufficiently adhered to a sufficiently gel to generate measurable substrate deformations and thus to calculate traction forces. Cells will embedded in a collagen gel containing poly-lysine to increase cell attachment strength. The collagen gel will be formed by mixing 1.55 mg/ml collagen type I (BD Biosciences) in 100 mM HEPES buffer at pH 7.3 and placing this solution at 37¿C for 30 minutes to gel. Another gel solution will be made containing the same concentration of collagen (1.55 mg/ml) in the BDM-containing heart buffer solution plus 2.5% 500 nm diameter fluorescein- coated polystyrene beads (Polysciences), 0.05 mg/l poly-L-lysine-succinimide (sigma) and a myocyte suspension of 10,000 cells/cm2 based on the gel surface area. This solution will be pipetted on top of the original solution and allowed to sit at room temperature for 30 minutes before placing at 37¿C for 30 minutes. This low-temperature gelation allows the cells and beads to settle to the lower surface along one plane to facilitate microscopy and analysis. When the gel has polymerized, it will be washed 3 times with Tyrodes solution containing 2 mM calcium and allowed to sit in that solution until the cells begin to contract. C3.1.g. Myocvte Micropatterninci. We have developed two methods for micropatterning deformable membranes for engineering patterned myocytes. The protocols combine photolithographic, microfluidic and micro-grooving techniques, to micropattern cell adhesion sites on elastic membranes, with cardiac myocyte and fibroblast isolation and culture. The resulting in vitro models of cardiac tissue mimic important structural and functional aspects of native ventricular myocardium, and show improved preservation of cell-shape, alignment and connectivity, while providing control over cell-matrix adhesion and mechanical environment (e.g. application of stretch). Both of these approaches are described in detail in a new article in press in Nature Protocols (Camelliti et al., 2006). The original microfluidic patterning technique (also known as microcontact printing) uses flat silicone rubber membranes, onto which stripes are deposited that consist of extra-cellular matrix proteins to guide cell attachment (Gopalan et al., 2003). This technique is shown in Fig. 12A and involves a reusable microstructured silicone rubber stamp, which is cast from photolithographically etched electronics-grade silicon metal wafers. The stamp is manually sealed onto the target membrane, forming microfluidic channels for spatially-restricted application of matrix protein solution. After drying of the solution, the stamp is carefully removed, leaving a patterned growth substrate on the membrane. It is possible to double-print structures, for example to create regularly intersecting patterns of parallel lines, thereby transforming pseudo-ID structures (lines of cultured cells) into a pseudo-2D representation of tissue (grids). Isolated cardiac cells have been successful plated onto such tracks, made of collagen. This procedure provides good spatial resolution and definition of spatial anisotropy, but results in sparse cultures. Microfabrication of deep microstructures is achieved by molding a replica of polydimethylsiloxane (PDMS) from etched silicon wafers. The mold is then used to create a pattern of collagen, laminin, fibronectin, or a mixture by microfluidics. After 5 minutes, the microfluidic mold is removed and excess matrix protein washed away, leaving the pattern on the substrate. Myocytes are then cultured onto the membrane or dish as described above. On the patterned substrata, cells adhere, spread, grow and take on an elongated tissue-like phenotype. Non-specific absorption of proteins from serum is minimal since the cells are serum starved during the stretching protocols (to prevent non-stretch-induced cellular growth). Cellular adhesion away from the 215 Knowlton, Kirk U. substrate patterns can also be reduced by modifying the hydrophobic surfaces with polyethylene oxide polymers (Li et al., 1996) or covalently coupled polyethyleneglycol (PEG). Spin negative photoresist le to UVthrough mask ¿Transparency mask ^Develop exposed photoresist Mlcropatterned SI wafer [Cast POMS mold PDMS main with micro-channels PDMS mold on membrane Fill channelswith collagen h arc In Incubator Remove PDMS mold Collagen tracks on membrane Cells on parallel collagen tracks POMS mold on membrane 'Fill channels with collagen 1 h3T*C in Incubator 1 Remove PDMS mold 40'C overnight Criss-cross collagen Plate cella tracks on membrane Cells on criss-cross A collagen .tracks Figure 12. A. Micropatterning procedures. B. Equibiaxial and non-equibiaxial cell stretcher assembly The second method for micropatterning involves fabricating grooves into the silicone membrane itself (Fig. 13). With this method, cells are more confluent on the membrane, and thus more protein is available for analysis from each stretcher. Photolithographic patterning of silicon wafers is carried out as described previously (Bhatia et al.,1998). Briefly, negative photoresist is coated on a silicon wafer, exposed to ultraviolet light (365 nm) through a transparency mask, and developed using high resolution printing of patterns with required width and separation between the parallel lines (10 u,m parallel lines and 10 UJTI spacing). Polydimethylsilioxane (PDMS) is prepared from a mixture of 2 liquid components (Sylgard 186 Kit, Dow Corning), poured onto developed wafer as a thin layer and after degassing, and cured. The resulting membranes are coated with the re uired ECMfor aligning Itie myocytes. Figure 13. Novel myocyte patterning assay for Membrane micropatteminn improved quantitative measurements of protein changes due to chronic cell stretching. A PDMS PDMS Mold mold is fabricated and used to make thin silicone membranes with the same alignment pattern. A collagen (or other substrate) layer is Silicone membrane applied to the surface, and myocytes applied to the membrane. The cells align with the membrane and take an elongated, rod-like Collagen coating phenotype, and stretch patterns (longitudinal and/or transverse) are then applied to these cell preparations. Myocytes align with pattern C3.1.h. Stretchers and Membranes. Silastic deformable membranes are prepared with a collagen type I and other substrate coatings similar to that used by Sadoshima et al. (Sadoshima et al.,1992). The cells (2 * 105 cells/cm2) are plated on patterned or unpatterned silicon membranes coated with collagen type-1 and equipped on equibiaxial (uniform) or non-equibiaxial (elliptical) stretcher devices as shown in Fig. 12B (Lee et al., 1996). The cells are serum-depleted for 24 hr followed by passive stretch for an additional 24 hr. Total RNA is extracted and subjected to RNA protection assay (RPA) using Direct RPA system (Ambion Inc.). Mouse BMP 216 Knowlton, Kirk U. coding sequences were amplified by RT-PCR and used as the template for a BMP riboprobe synthesis with mouse GAPDH as a control. Micropatterning allows aligned myocyte cultures to be subjected to non- equibiaxial stretch on the elliptical stretcher (Gopalan et al., 2003), which applies anisotropic strain to cells in a constant ratio (e.g. 2:1). If cells are patterned with their axis aligned with one axis of the elliptical stretcher, the ratio of principal strains is defined by the ratio of minor to major diameters of the ellipse. With these devices, the magnitude of strain is precisely controlled at any value and calibrated for each stretcher. C3.1.I. Cell Preparations for Histology. In order to evaluate the sarcomeric definition and organization in myocytes plated on gels of different rigidities, cells are fixed and stained for markers such as a-actinin at 48 hours and 7 days after initial plating. Cells are fixed by washing with cold (4¿C) PBS followed immediately by immersion in cold 0.75% glutaraldehyde for 20 minutes. Cells membranes are permeabilized with 0.1% Triton X100 (Sigma) for 5 minutes. Antibodies are diluted in 1% bovine serum albumin (BSA, Gemini) and added to the fixed cells for 60 minutes at room temperature. Secondary antibodies of tetra-rodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit (Jackson Immunoresearch, West Grove, PA) are diluted in 1% BSA and added for 30 minutes. Cells are imaged with a confocal microscope at NCMIR or by phase-contrast and epifluorescence using our Nikon Eclipse TE300 inverted microscope and Photometries Cascade 51 2X CCD camera and acquired using Metamorph software (Universal Imaging Corporation). In addition, cell area and aspect ratio (long axis/short axis) are computed by segmenting cells with ImageJ software (NIH). C3.1.I. Ratiometric Imagine] of Intracellular Calcium Transients. Isolated cells are labeled with Fura-2 (Grynkiewicz et al., 1985). Fura-2 calcium indicator (Molecular Probes) is diluted to a concentration of 5 uM in a high-calcium Tyrodes buffer (130 mM NaCI, 5.4 mM KCI, 2 mM CaCI, 1 mM MgCI2, 0.3 mM Na2HPO4, 10mM HEPES, 5.5 mM Glucose at pH 7.4) and added at room temperature for 30 minutes. Cells are then washed 3 times with fresh Tyrodes buffer and placed in Tyrodes buffer for the duration of the experiment. Cells are imaged using a Nikon Eclipse TE3000 inverted microscope and stimulated to beat using the same procedure as in the traction force measurements. A phase-contrast image of the resting myocytes is taken in order to constrain the analysis of the image. Using a Lambda DG-4 high-speed filter changer (Sutler Instruments), cells are illuminated with alternating 380 nm and 340 nm light and the fluorescence at 510 nm is imaged using a Photometries Cascade 51 2X CCD camera and acquired using Metamorph software (Universal Imaging Corporation). The filters are switched and a new image is acquired every 15 ms for 4.5 seconds (300 images). In order to calibrate the Fura-2 response, an equal amount of high-calcium Tyrodes buffer containing 20 uM calcium ionophore-4-bromo-A23187 (Molecular Probes) is added to saturate the Fura bound to calcium and images are acquired at 340 nm and 380 nm excitation/ 510 nm emission. The media is removed and media containing 10% ethylenediaminetetraacetic acid (EDTA, Sigma) is added in order to chelate all calcium and result in a baseline Fura fluorescent signal. Again, images are acquired at a 340 nm and 380 nm excitation/ 510 nm emission. Images are analyzed using custom macros programmed in Metamorph or ImageJ (NIH) to split the image sequence of alternating images at 340 and 380 nm excitation into 2 stacks, compute the ratio of those stacks and compute the average value of that ratio for each image inside the cell outline for manually outlined cell areas. The concentration of free calcium in the cell is then computed using the following formula: -R] max / where kd is the dissociation constant (0.14 uM), Q is the ratio of the total average fluorescence inside the cell outline at 380 nm excitation, R is the ratio of fluorescence at 340 nm excitation to the fluorescence at 380 nm excitation in each image, Rmin is the same ratio upon addition of EDTA and f?ma* is the same ratio upon addition of the calcium ionophore. C3.1.k. FRET Imaging of Intracellular Signaling Events. Neonatal cardiac myocytes are transfected with a recombinant FRET reporter, such as the A-kinase activity reporter AKAR2 (Zhang et al., 2005; Zhang et al., 2001) 1 day after isolation using FuGeneS transfection reagent (Roche Diagnostics) and imaged 2 days after transaction (-5% transfection efficiency). For adult cells we will used viral-mediated gene transfer. AKAR2 is a recombinant protein composed of the yellow (YFP) and cyan (CFP) mutants of GFP, a protein kinase A substrate, and a phosphothreonine-binding domain. PKA-mediated phosphorylation of AKAR2 increases FRET 217 Knowlton, Kirk U. from CFP to YFP, allowing real-time fluorescence imaging of endogenous PKA activity by the yellow/cyan emission ratio. Fixed myocytes expressing reporter are labeled for a-actinin to confirm normal cell morphology. Cells are washed with Hanks buffered saline solution with 20 mM HEPES buffer (pH 7.2) 20 minutes prior to imaging and kept at 35 degrees C with a stage heater (Carel) during imaging. Transfected myocytes are imaged on a Nikon Eclipse TE300 inverted microscope equipped with a 60X Plan Apochromat objective, Photometries Cascade 512F CCD camera, and MetaFluor 6.2 software (Universal Imaging Corporation). Imaging is performed using a 430/25 nm excitation filter (for CFP) and simultaneously recording CFP (470/30 nm) and YFP emissions (535/30 nm) with a DualView emission splitter (Optical Insights; Chroma filters). Images are acquired with 1 s exposure every 5 s. To obtain emission ratio time-courses, YFP or CFP emission intensities for each image are averaged over a region of interest, background subtracted, and then yellow/cyan emission ratio is calculated, normalized by the ratio before reagent application. For emission ratio images, a similar approach is used on a pixel-by-pixel basis with a 5x5 pixel median filter using MetaMorph software (Universal Imaging Corporation). C3.2. Isolated Murine Trabeculae and Papillary Muscles C3.2.a. Muscle Isolation and Mounting. Male or female mice are anesthetized with Isoflurane. After cervical dislocation, the chest is opened and the heart is arrested by intracardiac injection of low calcium, high potassium cardioplegic solution. Then the heart is rapidly removed, cannulated, and perfused with modified Hepes buffered solution containing (mM): 137.2 NaCI, 15.0 KCI, 1.2 MgCI2, 2.8 mM Na acetate, 10 taurine, 1.0 CaCb, 10mM glucose, and 10mM Hepes in equilibrium with 100% O2. The right ventricle is opened and a right ventricular papillary muscle is dissected free by first cutting a small section of the valve, to which the muscle is attached by way of the cordea, and then cutting a small section of the septal wall, to which the muscle it attached at the base. Papillary muscles are chosen based on their geometry. Typically papillary muscles chosen for these studies are long, thin, and unbranched. Studies are not done on left ventricular (LV) papillary muscles because these muscles are thick and diffusion of oxygen and nutrients to the core of these muscles may be limited. Additionally, ANP and BMP induction may be effected by a decreased oxygen supply in LV papillary muscles and hence these specimens may not have a normal stretch induced response (Chen, 2005; Sabatine et al., 2004). The muscles are mounted in the cardiac tissue culture chamber, containing the same Hepes buffered solution used for the dissection. Muscles are mounted between a basket attached to a force transducer and actuator controlled micromanipulator, and a stationary titanium hook like extension. Oxygen is flow over the solution during the mounting procedure, to prevent hypoxia. All portions of the system, for experiments longer than 6 hours in time, are steam sterilized prior to each experiment. Additionally, all solutions are filter sterilized. The Hepes buffered solution is then exchanged for a modified M199 cell culture media containing (mM): 2.0 L- carnitine, 5.0 creatine, 5.0 taurine, 2.0 L-glutamine, 0.2% albumin, 100 IU/ ml penicillin, 0.1 mg/ml streptomyocin, 10mM Hepes, and 50 ug/ml of insulin in equilibrium with 5% CO2and 95% Oa.The temperature of the muscle chamber is maintained at 34¿C. The pH of the solution is maintained between 7.4 and 7.5. The modified Hepes buffered solution is exchanged for modified M199 media in steps, such that calcium concentration is slowly increased from 1.0 mM to 1.75 mM. Once the solution is replaced, the muscle is stimulated via the hook and a platinum electrode positioned within close proximity of the muscle's base. Muscles are left at slack length and stimulated at 0.2 Hz for one hour (Janssen et al., 1998). After the muscle has equilibrated, it is either stretched between 85-95% of Lmax, where Lmax is defined as the length of the muscle at which the muscle produces the greatest active force, or left at slack length for the duration of the experiment (2 hr, 5 hr, 12 hr). Muscle lengths are acquired with an LVDT in parallel with the micromanipulator. The surface of each muscle can be marked with titanium dioxide markers, such that local muscle deformations can be observed during the experimental procedure. Muscle dimensions are acquired by video capture, calibrated with a phantom of known dimensions placed at the focal plane of the specimen. C3.2.b. Mechanical Testing. Right ventricular papillary muscles are mounted between a stationary hook and an Isometric Harvard Apparatus force transducer (724490), which is capable of measuring force ranges between 0-0.5g and has an accuracy of ¿1% (<1 mg). The Harvard Apparatus force transducer is attached to a Newport 460P series, high precision, linear modular ball bearing stage, whose position in the x/y/z direction is controlled by a micromanipulator, which is connected to a Newport motorized actuator (CM-12CC), having a resolution less than 100 nm, an incremental motion less than 500 nm, and a speed range between 50-500 218 Knowlton, Kirk U. Urn/sec. In order to measure muscle lengths and distances by which the muscle is stretched with a precision on the order of 1 |xm, a LVDT (Omega LD310-10) having an accuracy of 400 nm and a linear range of 20 mm, is mounted in parallel with the actuator. Using this experimental setup, uniaxial muscle forces are recorded while pacing the muscle at 1 Hz and during continuous stretching of the muscle up to Lmax. Muscle lengths are simultaneously acquired and local deformations of the muscle are imaged by a CCD camera (COHU Inc.). Digital video recordings of muscle deformation are synchronized with acquired force data. Lagrangian uniaxial strain measurements (E=1/2(A2-1), where Ais the stretch ratio) are calculated with respect to the slack length of each muscle. Lagrangian stresses are calculated by dividing acquired force data by the initial cross sectional area of each muscle. C3.2.C. Real-Time PCR Procedures. At the end of each muscle experiment, the muscles are immediately submerged in RNA later solution, which functions to stabilize mRNA in intact tissue samples and contains RNase inactivating reagents (Quiagen). Right ventricular papillary muscles are completely isolated from their attachments, the valve and septal wall. Then the muscles are homogenized and RNA is extracted from these tissues, using Quiagen's RNeasy Micro RNA purification kit. Given that total RNA extraction from these small specimens is low (-10 ng/|jJ) and that DNase treatment has been shown to degrade and or destroy nucleic acid extracts, DNase treatment is not applied to RV papillary muscle specimens (Jemiolo and Trappe, 2004). Reverse transcription (RT) is accomplished using the Invitrogen Super Script III cDNA synthesis kit, which is optimized for low inputs of RNA. Finally, quantitative PCR is performed using an Applied Biosystems ABI 7700 real time thermal cycler, AB Taqman Universal Master Mix with UNG, with AB pre-made primers and taqman probes for ANP, BMP, and GAPDH. ANP and BNP gene expression is normally induced by increased mechanical loads in isolated cardiac myocytes as well as intact cardiac tissue (Ruwhof et al., 2000). GAPDH housekeeping gene expression does not change in these experiments. For each amount of RNA tested, duplicate Ct values are obtained and averaged. Then the AACt method is applied to the averaged Ct values obtained, in order to quantify the fold change of ANP or BNP relative to the unstretched control samples. Within each gene expression assay, appropriate controls are also included. No RT controls are performed for each amount of RNA, testing for any genomic DNA contamination. Also negative controls, where no cDNA is added to the reaction, are performed in order to test the purity of the enzyme master mix as well as the primer and probe mixture. Data are presented as mean ¿ 1SEM. Because mRNA concentrations are log-normally distributed and require nonparametric statistics, a two group t-test is performed on the ACt values or the raw output data obtained from the thermal cycler, between stretched and unstretched experimental groups. Significance is set at the P < 0.05 level, with appropriate corrections for multiple comparisons. C3.2.d. Antibody Staining and Confocal Microscopy. Papillary muscles that are not used for gene expression analysis are stretched to some known % stretch and fixed in 2% PFA (para-formaldehyde) diluted in 50% PBS (phosphate-buffered saline) and 50% modified Hepes buffered cardioplegic solution. After 10-20 minutes, the solution is replaced with 4% PFA in 100% PBS. Muscles are allowed to fix for an additional 10-20 minutes and then transferred into Tissue-Tek O.C.T. compound. They are frozen on dry ice and stored at -80¿C. Using a cryostat, tissue samples are then sectioned (10-15 u,m sections). For ANP protein analysis, tissue cross- sections are prepared, while for titin epitope, staining longitudinal sections are prepared. After a series of blocking and washing steps, tissue sections are then stained with appropriate antibodies (e.g. IgG antimouse ANP antibodies are used to look at ANP protein expression in stretched and unstretched specimens, IgG monoclonal anti-mouse a-actinin (Sigma) antibodies stain for sarcomeric z-disks, monoclonal antimouse IgM 9D10 antibodies stain for the PEVK region on titin (Hybridoma Bank), and polyclonal anti-goat IgG telethonin antibodies stain for Tcap (Santa Cruz Biotechnology), a protein adjoined to the N-terminal end of titin). Appropriate secondary antibodies conjugated to either Alexa 568 or Alexa 488 were also used. For titin epitope staining procedures, the tissues were double stained with either a-actinin and Tcap or a-actinin and 9D10. A Biorad confocal microscope is used to image the sections with filter sets appropriate for the excitation of Alexa- 568 and Alexa-488 simultaneously. Distances between titin epitopes and the z-disk were determined using FFT analysis as we reported earlier for skeletal muscle fibers (Shah et al., 2004). 219 Knowlton, Kirk U. C3.3. Isolated Perfused Whole Mouse Heart Preparations C3.3.a. Isolated Mouse Heart Preparations. Mice are administered an intraperitoneal injection of heparin(100 USP Units), anesthetized with isoflurane and sacrificed by cervical dislocation. The heart is rapidly excised, washed in cold cardioplegic solution, and the aorta cannulated on a 20-gauge stainless steel cannula. The remaining tissue surrounding the aorta is tied closed using 5-0 suture to seal all vasculature to the heart. The heart is mounted on a retrograde Langendorff perfusion apparatus (Radnoti). The coronaries are perfused with an oxygenated modified Krebs-Henseleit solution: 24.9 mM NaHCOs, 1.2 mM KHaPO^ 11.1 mM dextrose, 1.2 mM MgSO4, 4.7 mM KCI,118 mM NaCI, and 2.55mM CaCI2 (Grupp et al., 1993). The perfusate is bubbled with 95% O2-5% CO2 gas, maintained at 35-37¿C, and a flow rate of 1-2 ml/min is achieved by applying a constant pressure of 70 mmHg. The coronaries are cleared and the heart is allowed to beat freely and equilibrate for 15 minutes. Surface EGG electrodes are set in place and recordings are taken throughout the experiment. 95% 02 / 5% COj The heart is paced with a digital stimulator (DS8000, World Precision Instruments). It is paced epicardially from either Retrograde Perfuston the atria or ventricle with a bipolar platinum electrode at a Reservoir constant current two times threshold. For isovolumic Langendorff preparations, a fluid filled balloon is inserted into Antarograde the left ventricle through the mitral valve. The balloon, Perfusion Reservoir consisting of plastic wrap at the end of a 20-gauge blunt needle, is connected to a syringe infusion pump and inline with a fluid-filled pressure transducer. The balloon is inflated Compliance Chamber and deflated to an EDP of 30 mmHg to precondition the Moan Aortic P Measurement tissue. For the working heart preparation, the heart contracts Outflow Resistance against a resistive-compliant network consisting of a chamber f. Cardiac Output with an air bubble and a flexible tube with an adjustable clamp (Fig. 14).The heart is kept immersed in warm (37¿C) Collection Reservoir KHS throughout the experiment except when marker deformation is being videotaped. The pressure in the aortic Right Atrtal outflow line is monitored using a transducer (Millar Pacing Loads Instruments, Houston, TX). Video Camera Figure 14. Schematic of isolated ejecting heart apparatus in the VCR Core A lab. (From Karlon ef a/. (Karlon et al.,2000)) C3.3.b. Non-Homogeneous Epicardial Strain Analysis. Non-homogeneous epicardial strain distributions are mapped in the isolated mouse heart using high-resolution optical imaging of 50-100 small titanium dioxide markers arrayed on the epicardium with a short-bristled brush. In addition to LV strains, we have also used these methods in our lab to map regional distributions of septal strains in isolated working and non-working mouse hearts (Karlon et al.,2000) and to reconstruct right ventricular strains during systole (Lorenzen-Schmidt et al.,2005). A bipolar stimulating electrode will be used to pace the heart from the right atrium and a high- fidelity Millar pressure-volume catheter will be used to measure ventricular volume and pressure. A high- resolution CCD camera will be used to image the epicardium during isovolumic or ejecting contractions at full range of end-diastolic volumes. Markers will be tracked to sub-pixel accuracy throughout the cardiac cycle using the object tracking tools of the MetaMorph (Universal Imaging) software package. The markers are mapped to a bicubic Hermite finite element mesh and a least-squares fit of marker displacements is used to compute two-dimensional non-homogeneous strain fields using software developed in Dr. McCulloch's laboratory (Karlon et al.,2000; Mazhari et al., 1998). Briefly, the epicardial surface is modeled with 6-8 bi-cubic Hermite prolate spheroidal finite elements, and two-dimensional reference marker coordinates measured from the video images are projected on to the surface. The deformed coordinates are then projected on to the same material points in the models, which are then refitted by least squares resulting in a parametrically deformed mesh from which regional strains can be interpolated at each frame. This approach has also been used in conjunction with optical mapping of electrical activity in the isolated perfused rabbit heart (Sung et al.,2003). In addition to computing isovolumic strains (which are substantial), it is possible to obtain strains representative of ejection phase wall motions in the non-working heart, by using a reference state at an end-diastolic frame at mid-high ventricular volume and a deformed state at an end-systolic frames at lower ventricular volume. These 220 Knowlton, Kirk U. techniques can all be applied to the right ventricle as well as the left, which will be important for Project 2 (Chen). C3.3.C. Optical Mapping of Action Potential Propagation. A 0.8 ml bolus of the voltage-sensitive dye di-4- ANEPPS (25 uM) is injected into the perfusion line. The ventricular epicardium is illuminated by two 470 nm wavelength, 3.6-W 40-LED cluster bulbs (Ledtronics, Inc.). Fluorescence emission is passed through a >610 nm high-pass filter, focused with a fast 50 mm lens (1:0.95, Navitar) and recorded by a 12-bit charge-coupled- device (CCD) camera (CA-D1-0128T, Dalsa). Images of a 9x9 mm2 field of view are acquired at 950 frames per second and a spatial resolution of 64x64 pixels. The time series of fluorescent images provides temporal intensity signals at each pixel. Each signal is normalized with respect to baseline intensity (AF/F) and inverted to better represent a cardiac action potential. Spatial phase-shift and temporal median filtering (Sung, 2001) are implemented to reduce noise in the signal. Activation time is determined as the time of maximum slope (dF/dt) during the action potential upstroke. This provides an activation map over the ventricular epicardium showing the rate and path of action potential propagation. Spatial gradients of activation time are used to calculate local apparent conduction velocity vector at each pixel (Bayly et al., 1998). Additionally, the curvature of the activation wavefront is calculated as the spatial gradient of the normalized conduction velocity vector field. Time of repolarization is calculated by identifying the peak signal and determining when the action potential has recovered 20%, 50%, and 80% back to baseline. Action potential duration (APD) is then defined as the time difference between repolarization and activation. APD dispersion is determined as the standard deviation of APD over the surface of the heart. Attenuation of motion during optical mapping studies is necessary for the analysis of repolarization. Modified Krebs-Henseleit solution with 1 mM CaCb and 15 mM of the electromechanical uncoupler 2,3-butanedione monoxime (BDM) is perfused during data acquisition to eliminate motion artifact. However, it has been demonstrated that BDM prolongs action potential duration and slows conduction velocity in the isolated murine heart (Baker et al., 2004). We have recently developed a combination of ratiometry and a motion tracking algorithm to eliminate motion artifact without the use of chemical uncouplers. Since the emission spectrum of the potentiometric dye is wavelength shifted with altered membrane potential, upright and inverted optical action potentials can be recorded at green (560 nm) and red (620 nm) wavelengths respectively. A wavelength splitting optical setup allows us to record fluorescence emission at low and high wavelengths simultaneously using a single CCD camera, a technique originally applied to ratiometric microscopy (Kinosita et al., 1991). Image registration and ratio calculation of the two wavelengths amplifies the magnitude of the recorded optical action potential and corrects for differences in pixel intensity values due to heterogeneous dye loading or uneven excitation lighting. To correct for myocardial motion during contraction, a Lucas-Kanade optical flow algorithm is used to compute a vector field of frame-to-frame pixel displacements that is used to perform a non- rigid sub-pixel resolution transformation of deformed frames back to the reference configuration. Combined, these techniques effectively correct for tissue displacement and local motion artifact in the optical recordings. C3.3.d. Programmed Stimulation. Restitution kinetics and vulnerability to arrhythmia are assessed in the isolated mouse heart by programmed S1-S2 stimulation protocol. The isolated heart is paced from the left ventricular epicardium via a bipolar platinum electrode with a digital stimulator (World Precision Instruments, DS8000). The ventricle is paced at a basic cycle length of 200ms (S1-S1) for >20 beats followed by a premature stimulus (S2) (Baker et al., 2000; Lerner et al., 2000). The S1-S2 cycle length is decreased until the effective refractory period is reached, i.e. when the S2 stimulus no longer induces an action potential. During this protocol, optical mapping is used to analyze action potential morphology (amplitude and duration) and conduction velocity of the S2 induced action potential (Knollmann et al., 2006). The relationship between these measurements and the S1-S2 diastolic interval can be used to assess the restitution kinetics of the mouse heart. Premature action potentials during short S1-S2 cycle lengths can induce arrhythmias in susceptible media. The arrhythmia vulnerability of transgenic hearts can be assessed by the incidence of ventricular tachycardias during this protocol (Lerner et al., 2000). Additionally, burst pacing or a train of several high- frequency S2 stimuli can induce arrhythmias in the isolated heart (Fig. 15). C3.3.e. Electrical Space and Time Constants. The effective space and time constants directly aggregate myocardial electrical resistance and capacitance and are measured in the isolated mouse heart using similar methods to those of Poelzing et al. (Poelzing et al., 2005). A 125-micron diameter Teflon-coated platinum 221 Knowlton, Kirk U. unipolar electrode was used to pace the mid-lateral LV free wall. After steady-state pacing from the electrode (100 paced beats), a 1-mA cathodal stimulus was delivered for 250 ms, 35 ms after the final drive train pacing stimulus, during the refractory period of the previous action potential, resulting in a cathode-break stimulus upon release of the 250 ms pulse. Signals are normalized by baseline action potential amplitude so that the tissue stimulus response could be compared across space. The common mode signal (mean signal distal from the stimulus