Institute of Molecular Cardiology

by Tang, Xian-Liang — last modified Oct 17, 2011 11:47 AM

Biochemistry/Molecular
Biology Laboratories

I. Introduction

In 2001, the University of Louisville established the Institute of Molecular Cardiology in order to facilitate and foster cardiovascular research. Under the direction of Dr. Roberto Bolli, the Institute of Molecular Cardiology has grown considerably over the past decade, and currently includes a large group of investigators, with 21 faculty members and more than 60 staff members, Research Associates, Technicians, Postgraduate Fellows, etc. The work performed in the Institute has made significant contributions in various fields, particularly myocardial ischemia/reperfusion injury, cardioprotection, environmental cardiology, diabetes, and postinfarction LV remodeling and heart failure. The productivity of the Institute is documented by the large number of peer-reviewed publications (since 2001, 328 full manuscripts) published by institute members. Detailed information can be found at http://louisville.edu/medschool/cardiology/Publications.html

1. Mission

The mission of the Institute of Molecular Cardiology is to coordinate the cardiovascular research program at the U of L School of Medicine in order to (i) facilitate collaboration and synergy among investigators, (ii) utilize our cardiovascular resources optimally and efficiently, (iii) strengthen, expand, and integrate basic and clinical research programs, (iv) facilitate rapid translation from bench to bedside, and (v) provide institutional support to investigators engaged in cardiovascular research.

2. Faculty

The Institute of Molecular Cardiology includes a large multidisciplinary team of 22 faculty members who have different but complementary backgrounds and are highly motivated, collegial, and enthusiastically totally committed to excellence in cardiovascular research:

Oleg Barski, Ph.D., Assistant Professor of Medicine	Aruni Bhatnagar, Ph.D., Professor of Medicine
Roberto Bolli, M.D., Professor of Medicine, Physiology, and Biophysics	Chuanxi Cai, Ph.D., Assistant Professor of Medicine
Daniel Conklin, Ph.D., Associate Professor of Medicine	Michael Flaherty. M.D., Ph.D., Assistant Professor of Medicine
Yiru Guo, M.D., Associate Professor of Medicine	Wysoczynski,Marcin Ph.D., Instructor in Med-ardiology
Jia-Qiang He, Ph.D., Assistant Professor of Medicine	Bradford Hill, Ph.D., Assistant Professor of Medicine
Kyung Hong, Ph.D., Assistant Professor of Medicine	Steven Jones, Ph.D., Associate Professor of Medicine
Qianhong Li, M.D., Ph.D., Assistant Professor of Medicine	Detlef Obal, M.D., Assistant Professor of Anesthesiology
Timothy O’Toole, Ph.D., Assistant Professor of Medicine	Gregg Rokosh, Ph.D., Assistant Professor of Medicine
Matthew Spite, Ph.D., Assistant Professor of Medicine	Sanjay Srivastava, Ph.D., Associate Professor of Medicine
Xian-Liang Tang, M.D., Associate Professor of Medicine	Srinivas Tipparaju, Ph.D., Assistant Professor of Medicine

3. Space

The Institute of Molecular Cardiology occupies ~50,000 sq. ft. of space in three buildings: the Medical-Dental Research Building ((4,320 sq ft.), the Donald E. Baxter Biomedical Research Building (Baxter I, 7,052 sq. ft.), and the Delia B. Baxter Biomedical Research Building (Baxter II, 37,850 sq ft.). Surgical procedures and studies of cardiovascular physiology are performed in the Medical-Dental Research Building, whereas biochemical and molecular studies are conducted in the two Baxter buildings. All of these buildings are in close geographic proximity. This, together with the sharing of resources and projects among faculty, reinforces the integrated work in myocardial ischemia and stem cell biology that represents the overarching focus of the Institute of Molecular Cardiology.

The Donald E. Baxter Building is a 120,000 sq. ft. state-of-the-art research facility; the Institute of Molecular Cardiology occupies half of the first floor. This building contains the Transgenic Mouse Core facility, the Proteomic Core, and the Institute for Cellular Therapeutics. The Delia B. Baxter Building contains 130,000 sq. ft. of space featuring 48 laboratories. The Institute of Molecular Cardiology occupies the entire fourth floor, 3/4 of the third floor, half of the second floor, and 3 laboratories on the first floor of this Baxter building, corresponding to ~37,850 sq. ft. of space. This building includes a large animal vivarium of 19,000 sq. ft. which is designed to house up to 35,000 mice in both conventional and limited-access sterile barrier conditions (in this facility, “conventional” housing includes individually-ventilated cage rack systems and microisolator techniques).

Medical Dental Research Building (MDR)

	Donald E. Baxter Biomedical Research Building (Baxter I)

Delia B. Baxter Biomedical Research Building (Baxter II)

4. Support

The Institute is supported in part by University institutional resources that are targeted to recruitment of faculty and overall administrative support, providing a clear testimony to the commitment of the School of Medicine to the development and success of cardiovascular research. The vast majority of the expenses of the Institute, however, are supported by external competitive grants. Numerous NIH and AHA grants are currently funded within the Institute of Molecular Cardiology, including a Program Project grant (“Protection of the Ischemic Myocardium”, led by Dr. Roberto Bolli), a U24 grant (Consortium for preclinicAl assESsment of cARdioprotective therapies [CAESAR], led by Dr. Roberto Bolli), and a COBRE grant (Center of Excellence in Diabetes and Obesity, led by Dr. Aruni Bhatnagar). The goal of these multi-investigator programs is to examine novel therapies to protect the heart from ischemia/reperfusion injury and the effects of obesity and diabetes on cardiovascular disease.

5. Collegiality

The entire Institute of Molecular Cardiology is predicated on the basic principle of team work and collegiality. Candidates for recruitment to the faculty are prospectively selected on the basis of their willingness to be collegial. Candidates who are not interested in collegial work are not recruited. All faculty members are expected to work with other members in joint projects and to assist colleagues who need technical assistance, reagents, equipment, or input and advice. Virtually all research projects conducted in the Institute represent collaborations among faculty members. The Institute holds regular (weekly) faculty meetings in which research in progress is presented and discussed on a rotating basis by all Institute members and every faculty member or trainee is expected to present his/her data for collective discussion. At these meetings, all participants (faculty and trainees) provide input, feedback, suggestions, critiques, and insights. This is how collegiality is achieved within the Institute. The real dimensions of this collegiality can be appreciated by examining the joint publications, joint grant proposals, and joint projects among Institute members.

The very existence of three multi-investigator projects (the current program project, the U24 Consortium, and the COBRE grant) is an eloquent testimony to the depth and intensity of the collaborative work among members of the Institute. Without a genuine team spirit, none of these three multidisciplinary programs would have been possible or even thinkable. It is precisely this culture of collaboration that has enabled the Institute to grow to its present status from a situation 15 years ago in which virtually no basic research in cardiovascular medicine was being done in Louisville. The Institute members are proud of this culture. Unanimously, they are unwaveringly committed to preserving and augmenting this team spirit, which we regard as our major strength.

It is this culture that has enabled the current Program Project to develop, and it is this same culture that will enable the present proposal to succeed.

II. Animal Laboratories

1. Space

The institute of Molecular Cardiology has large, well-equipped animal laboratories, located on the first floor of the Medical Dental Research (MDR) Building. The space is approximately 4,320 sq. ft. and includes a room for fluoroscopy and acute surgery, five rooms for training and monitoring of conscious animals and for acute experiments, a lab for Langendorff preparations, a room for echocardiography and hemodynamic studies, and a room for the radioactive counter and storage. In addition, the Research Resources Center, which is located on the floor below the laboratory, is a state-of-the-art AAALAC-approved facility containing two fully-equipped, recently-built operating rooms (219 square feet each) dedicated exclusively to sterile surgery, two scrub rooms (48 square feet each), prep rooms, and recovery rooms. Since the laboratories of the Institute of Molecular Cardiology are located on the floor above the animal vivarium, animals can be easily transported back and forth from the vivarium to the labs and vice versa. Several full-time technicians and postgraduate fellows are available for assistance with the studies.

2. Major Equipment

Major equipment available in the animal laboratories includes a clinical Siemens Arcadis Avantic high-end mobile fluoroscope for coronary cineangiography, a PID Detection System for accurately detecting low level volatile organic compounds (VOCs) and toxic gases in air samples, a NOGA cardiac navigation system for electroanatomical endoventricular mapping (WBI, CA), a blood gas machine (model BAL 555, Radiometer Copenhangen, Denmark), three Gould 8-channel Thermal Array Recorders (model TA6000), a Gould 4-channel direct writing chart recorder (model RS 3400), all of which are equipped with signal conditioners and amplifiers; three 8-channel Doppler panels with modules including blood pressure, ECG, 20 MHz Doppler velocity, and 10 MHz Doppler displacement and two 3-channel Doppler panels with modules including blood pressure, ECG, and 20 MHz Doppler displacement; four oscilloscopes (3 model Heath 4554 and 1 model Hameg 205-3); two Powerlab data acquisition systems (model 4/30, AD Instruments, Colorado Springs, CO); a veterinary anesthesia ventilator and vaporizer system for large animals (VetLand ventilator EX3000, Louisville, KY); six Harvard Rodent Ventilators (model 683); four Harvard syringe pumps for withdrawal or infusion (2 model 22 and 2 model 944); a Valleylab electrosurgical unit (model Surgistat); a foot controlled Assi Polarmate bipolar coagulator (model AC-1905), two defibrillators (1 model Hewlett Packard 43130 and 1 model ISD); a gas anesthesia machine (model 21DC); three electrical stimulators (2 model Astro-Med/Grass SD9 and 1 model Bloom DTU-101VT); four temperature probes [2 Digi-Sense Type T Thermocouple Thermometer with a 23G implantable Teflon insulated probe (model 91100-20) and 2 Yellow Springs Instruments series 400 with relative Telethermometer boxes (model 43TA)]; two high-fidelity Millar pressure transducers; six Konigsberg solid-state pressure transducers; a Digi-Med BPA blood pressure analyzer; a Kinematica AG Polytron tissue homogenizer (model PT300); three surgical lamps (Polaris, AMSLO); a centrifuge; three refrigerator/freezers; a hematocrit centrifuge (Readacrit); three water baths with circulating pumps (1 model Haake W13, 1 model Neslab Exacal EX-200 and 1 model Isotemp 202), an AB54 S analytical electronic balance (Mettler Toledo, Switzerland), a Nikon camera with a Micro-Nikkor 105 mm f2.8D AF lens (Nikon, Japan), a complete IonOptix myocyte cell shortening fluorescence photometry calcium imaging system with imaging and IonWizard analysis software, a Warner perfusion valve controller and heater/flowmeter, a modified Langendorff setup for myocyte isolation, Radnoti isolated organ bath system, a dual impedance stimulator (Harvard Apparatus), a cell incubator, a laminar flow hood, a spectrophotometer, and three surgical tables.

3. Mouse laboratory

All of the mouse surgery is conducted in five dedicated rooms (two ~600 sq. ft. rooms and three ~250 sq. ft. rooms) which are routinely cleaned and sterilized. The room provides enough space for five surgeons to operate simultaneously.

The lab possesses extensive equipment for virtually any type of cardiovascular research in mice. Major equipment specifically available for mouse surgery includes microsurgical loupes (Designs for Vision, TN), four rodent ventilators (Harvard Apparatus, MA), four MiniVent ventilators (850 Hugo Sachs Elektronik, March-Hugstetten, Germany), a multiple-channel thermal array recorder (Gould Instrument Systems, OH), an Omni-Pacer LC4 motor-driven treadmill (AccuScan Instruments, Inc., Columbus, OH) with an adjustable belt speed (0-100 m/min) and shock bars with adjustable amperage (0-2 mA), an inverted tissue culture microscope (Nikon, Model SMZ-U) for taking pictures of mouse heart slices to measure infarct size, two Polar-Mate bipolar coagulators and Jewelers bipolar forceps (ASSI), four Type T thermocouple thermometers (Cole-Parmer), digital hygrometer/thermometers (Fisher Scientific), two oxygen flowmeter regulators (Fisher Scientific), a surgical light (Designs for Vision Inc., Ronkonkoma, NY) for microsurgery, a halogen bright spot surgical light (Burton Medical Products, Inc., Chatsworth, CA), four electrical mini heating pads, 10 cm dissecting scissors, curved-locking micro-needle holders, iris scissors, vessel dilators, extra-fine Graefe forceps, micro-mosquito forceps, Olsen-Heger needle holders, autoclip applier and remover, wood clips, and a compact hot bead sterilizer (model FST 350) for surgical instruments.

4. Echocardiographic Equipment

The Institute of Molecular Cardiology houses a state-of-the-art VisualSonics Vevo 2100 high frequency, high resolution (30 micron) digital imaging ultrasound system (VisualSonics, Inc.) equipped with 24 MHz and 38 MHz Microscan Transducers and with linear array technology for B-mode and M-mode imaging and color Doppler mode scanning. In addition, the Institute houses also a VisualSonics Vevo 770 High Resolution In Vivo Imaging System with similar capacity. This system is a non-invasive ultrasound-based method for viewing extremely small physiological structures in mice. The system images living tissue and blood flow with a spatial resolution down to 30 microns, the highest resolution available in real-time today. Specifically, the Vevo 770 allows: i) in vivo visualization of embryonic stage (E5.5) through adult mice in real-time; ii) comprehensive real-time cardiovascular imaging (M-mode, 2D, Doppler echocardiography) with anatomical and physiological evaluation of the developing and adult mouse heart, cardiac chambers, and associated major vessels; iii) ability to visualize ultrasound-guided needle injection and extraction; iv) high-resolution 3D rendering of areas of interest; v) microcirculatory and cardiovascular blood flow assessment; and vi) export of all image and analysis data in PC and Macintosh-compatible formats and offline data analysis. Also, the system permits in vivo micro-imaging using targeted and untargeted contrast agents. Untargeted microbubbles allow image enhancement of the blood pool and quntification of in vivo vascular perfusion in the heart and other tissues. Targeted, ligand-directed contrast agents can bind to a variety of biomarkers, thereby quantifying the target biomarker, including markers of inflammation and angiogenesis. The accompanying Integrated Rail System and Mouse Handling Table ensure that the mouse is maintained in the most stable and stress-free state during imaging. This imaging system offers the highest resolution real-time visualization currently available and as such will have multiple capabilities for both anatomic delineation and physiological characterization of a variety of organ systems including the heart, kidneys, liver, and vasculature, and pathologies such as atherosclerosis. The system also offers the unique capability for micro-imaging of tissues and vasculature using ultrasound contrast agents, 3D rendering of complex anatomical structures, and disease interventions with ultrasound-guided injection of drugs, growth factors, or other therapies.

The animal laboratory houses a state-of-the-art HP SONOS 7500 Ultrasound echocardiography system equipped with QuickTouch User Interface, Transthoracic Live xPlane imaging, which displays two high-resolution views of the heart simultaneously, giving you more information on which to base your diagnostic decisions. xMATRIX Transducer Technology and xSTREAM 3D Architecture's multidirectional beam steering enable real-time transthoracic Live xPlane imaging. Three different ultrasound probes (S8, S12, and L12-5), each of which operates at different wavelengths, offer the investigators the flexibility to image at different depths. Furthermore, a dedicated continuous wave Doppler probe allows flow imaging with greater accuracy. These features are extremely helpful for structural as well as functional evaluation of rodent hearts. Despite a very small depth, the walls and the valves of the heart in small animals can be visualized very well with this system, and the acquired data are optimal for various analyses (area, volume, flow, distance, slope, etc.). Image resolution is further enhanced by the high-definition zoom that enables precise measurement of various two-dimentional (2-D) and Doppler parameters by the investigators.

The HP SONOS 7500 system is equipped with several storage and analysis features that allow the investigator to measure distance, area, circumference, and 2-D volume. Volume/flow measurements can be performed easily and the user can define calculations. The images are viewed on a single 15-inch, high resolution, non-interlaced monitor and the system supports full range of scanhead technologies and applications. The ‘digital video streaming’ (DVS) feature of this machine allows the investigators to store and review images as digital frames or video clips. Real time bi-directional compression greatly enhances real-time analysis of data. Combined together, these techniques generate virtually artifact-free high-definition images at a near- or far-field. The HP SONOS 7500 system is also connected via a DICOM-compliant Server to a dedicated computer equipped with ComPACS Review Station (MediMatic). ComPACS Review Station is a complete multi-modality image management and reporting system designed to exploit advanced technologies to provide efficient and integrated scheduling, viewing, archiving, analysis and reporting of the acquired images. These features maximize the utilization of the system for data acquisition rather than analysis which can be performed off-line at the convenience of the investigator. In addition to digital imaging components, a VCR and two printers (color and black-and-white) are also available for cheaper storage of analog data for both off-line analysis and presentation. The color printer allows publication-quality reproduction of color images of intracardiac color Doppler flow signals. The black-and-white printer is primarily used for the generation of hard copies of online calculation frames for ready reference. The use of this black and white printer for day-to-day use lessens operational cost.

5. Hemodynamic Monitoring Equipment

The animal laboratory is equipped with an ARIA-1 single-segment pressure-volume (P-V) conductance system (Millar Instruments, Houston, TX) that can simultaneously and continuously measure high-fidelity left ventricular pressure and volume in the intact beating hearts of small animals such as mice. With the ARIA-1 system, pressure and volume signals can be plotted against each other in real time, generating the characteristic P-V loops that are an excellent illustration of the cardiac cycle in normal and diseased conditions. ARIA-1 P-V loops can be captured during physiologic, pharmacologic, and therapeutic interventions, allowing comprehensive evaluation of the fundamental mechanical properties of the heart. This ARIA-1 system provides the least invasive, most robust means for complete hemodynamic assessment in mice.

The ARIA-1 system consists of: (i) an MPCU-200 P-V signal conditioning hardware, (ii) two SPR-Series 1.4 and 2.0 French ultra-miniature P-V catheters, (iii) a 1.0 French PVR-1035 microtip ultra-miniature P-V catheter, iv) PVAN data analysis software, (v) a desktop pentium IV computer, and (vi) the data acquisition software (Chart, Powerlab Inc., ADInstruments). The MPCU-200 P-V signal conditioning hardware provides analog outputs of the time-varying ventricular pressure and volume signals for data acquisition. The combined pressure and volume signals generate P-V loops for the calculation of cardiac function and hemodynamic analysis in small animals. The MPCU-200 hardware has an unfiltered frequency response from 0 to 500 Hz with a high signal-to-noise ratio and combined pressure and volume signal conditioning. The conductance output is processed as the reciprocal of impedance to provide a true analog volume signal. The ultraminiature SPR-Series 1.0, 1.4, and 2.0 French P-V catheters (outer diameters of 0.33, 0.47 and 0.67 mm, respectively) have high frequency response (flat to 10 kHz), and via four platinum electrodes, are capable of measuring both left ventricular pressure and volume simultaneously from the intact, beating hearts of mice.

The data acquisition software (Chart, ADInstruments) can be used to simultaneously record pressure and volume, in addition to other physiological parameters such as ECG, blood flow, heart rate and intravascular blood pressure. The Chart data can be saved as text files and imported into PVAN Software for the calculation of end-systolic pressure/volume, end-diastolic pressure/volume, cardiac output, stroke volume, minimum/maximum dP/dt, and several other cardiovascular parameters. These raw pressure and volume data collected in text-files by the MPCU-200 unit and Powerlab are analyzed with the PVAN software (Millar Instruments, Inc.) that applies a variety of algorithms to the P-V data to calculate over 30 different hemodynamic parameters. PVAN contains an intuitive user menu offering easy access to a variety of displays and calculation tables for many different cardiovascular parameters. A saline calibration feature allows the user to obtain a value for the parallel conductance volume contribution of the myocardium. The ARIA-1 system is compatible with peripheral measuring devices with customizable graphical user interface, six channels of analog input, real-time display of P-V loops, real-time calculations using raw data (mean, max, min, derivatives, etc.), digital indicators (pressure, volume, heart rate, etc.), two-point calibration, and USB output.

The hemodynamic laboratory also has the accessories necessary for hemodynamic studies, including an operating table, several fine surgical instruments, two surgical lamps, and a vaporizer for the delivery of isoflurane anesthesia. In addition, the availability of the echocardiography machine in the vicinity of the hemodynamic set up offers the opportunity to accurately position the P-V catheter via ultrasound-guidance.

III. Biochemistry/Molecular Biology Laboratories

The Biochemistry/Molecular Biology laboratories of the Institute of Molecular Cardiology occupy over 27,000 sq. ft. of laboratory space on the first floor of the Donald E. Baxter Biomedical Research Building (Baxter I) and on the first, second, third, and fourth floor of the Delia B. Baxter Biomedical Research Building (Baxter II). Each floor is divided into two pods, and each pod consists of 6 laboratories for molecular/biochemical research, each occupying ~750 sq ft. Thus, over 27,000 sq. ft. of lab space is available to the Institute of Molecular Cardiology.

All laboratories and offices are supplied with computers and are wired for Ethernet T1 communications. Each laboratory is equipped with a fume hood and with equipment required to carry out all molecular and biochemical procedures, including benchtop microfuges and centrifuges, water baths, shakers, PCR thermocyclers, horizontal and vertical electrophoresis and blotting apparatus, balance, pH meter, refrigerators, freezers, homogenizers, sonicators, and spectrophotometers. In each of the pods, the remaining area accommodates core facilities (large equipment and culture facilities), a large walk-in cold room, 6 offices, and secretarial space that includes photocopier and FAX. In each pod, the core equipment area contains 2 Beckman J-25I and 1 Sorval RC26Plus superspeed centrifuges, two Beckman (LS-80 and Optima LE-80K) ultra centrifuges, two large Gyromax orbital incubator shakers, two SO-LOW Ultralow –80°C freezers, one Forma –152° C freezer, a Hewlett Packard Cobra II gamma counter, and a Hewlett Packard LS-6500 beta scintillation counter. Other core equipment includes BioRad 2800 binary HPLC, a Labconco Centritrap coldtrap and concentrator, and a BioRad 583 gel dryer. The central location of these laboratories in the two Baxter Biomedical Research Buildings facilitates use of the Proteomic, Pathology, and DNA Microarray Core facilities within the buildings. Other core facilities noted below are within the Health Science complex in adjacent buildings.

1. Major Equipment

The Biochemistry/Molecular Biology Laboratories in the Institute of Molecular Cardiology contain all of the equipment necessary to perform the proposed studies.
Major equipment available includes the following in Baxter I: a STORM 840 phosphorimager/fluorimager scanner and image analysis system (Molecular Dynamics) for the purposes of quantitative analysis of RNA dot blots, Southerns, Northerns, Westerns, a Molecular Dynamics PD-SI laser densitometer for quantitative autoradiography, a Sorvall J-25I superspeed centrifuge, two Beckman J-25I superspeed centrifuges, a Beckman LE-80 and an Optima LE-80K ultracentrifuges, a Beckman scintillation counter, a Plas Labs controlled atmosphere chamber (aerobic and anaerobic), two SO-LOW Ultralow –80°C freezers, two Gyromax orbital incubator shakers, a Monolight 2010 luminometer, a Perkin Elmer LS50B Luminescence spectrometer, a Pharmacia Biotech Ultrospec 3000 spectrophotometer, twelve room temperature and refrigerated benchtop centrifuges and microfuges, seven PCR thermocyclers, a ThermoScientific NanoDrop, two Millipore MilliQ water purification systems, two UV crosslinkers, a Kodak digital gel documentation system, and four hybridization incubators.

Additional major equipment housed in Baxter II includes the following: an Amersham/Biosciences Typhoon variable mode imager capable of luminescent, chemiluminescent, and phosphor imaging of protein, DNA, and RNA, two New Brunswick C25 incubator/shakers, two Beckman Avanti J-25 superspeed centrifuges, a Beckman J-E superspeed centrifuge, a Beckman OPTIMA LE-80K ultracentrifuge, two Millipore Milli-Q water purification systems, a FUJIFilm imagine machine, a ACUSON SEQUOIA ultrasound system, a multiscan spectrum, an AGILENT GC system, a fluorescence detector, a THERMO speedvac concentrator, a MILES SCI tissue processor, an eight-channel organ bath, a Leica auto rotary microtome, one AXON integrating patch clamp, one blood pressure acquisition system, one flexercell R strain unit, a Leica 3050 S cryostat, a Leica rotary microtone, a Packard Tricarb 2900TR liquid scintillation counter, six benchtop centrifuges (Heraeus Biofuge Pico), two refrigerated bench top centrifuges (Savant micro Speed fuge SFR13K & Sorvall Legend RT), a gel dryer (BioRad 583), several water baths, six full size refrigerators, four -20°C freezers, three -80°C freezers, an Eppendorf MasterCycler DNA thermal cycler, three BioRad MyCycler thermal cyclers, an Applied Biosystems Real-Time PCR System, an Applied Biosystems 7900HT fast real time PCR instrument, a ThermoScientific NanoDrop, Invitrogen and BIO-RAD complete 2D gel systems as well as multiple gel systems for agarose, mini-gels, and Criterion gels, a Gene Pulser Xcell System, a KODAK Image Station, a Packard Fusion fluorescent, luminescent, and UV/Vis microplate reader, a BioTek SynergyMx plate reader for absorbance, fluorescence and luminescence, a Tuner Biosystems luminometer, two spectrophotometers (an HP8453 and a Varian Cary 50 with Peltier system), a spectrofluorometer (Shimadzu RF5301 PC), a Micro ZMD Ion-Spray using Masslynx for interpretation, a Waters 1525 HPLC with a 2487 UV detector, a Waters 2690 HPLC with 996 photodiode array and 2410 refractive index detectors using Millennium 32 for interpretation, a BioRad Biologic HR FPLC, a Nikon Eclipse Se-300 inverted microscope with epifluorescence attachment that is dedicated to electrophysiology and a Hamamatsu photomultiplier C2761, a Tektronix 2213A Oscilloscope, an Adams/List P2P pipette internal perfusion system, a Bipolar temperature controller, a Harvard microscope stage incubator, a CCD camera to record the events, a Newport vibration-free table, a pClamp acquisition hardware and software, several Narashige micromanipulators, a system for Langendorff isolated heart preparations with a data acquisition system (ATMIO-16X and LabView Software), several pressure transducers and recording equipment, an Arkay blood chemistry analyzer, a Cobas Mira Plus clinical chemistry analyzer and Instech fiber optic oxygen meters.

The Institute of Molecular Cardiology has state of the art microscopic capability for the visualization of cells and tissues. These include: an OLYMPUS microscope equipped with evolution digital camera, a Nikon SMZ-U dissecting microscope, a Nikon ECLIPSE TE2000-U inverted microscope with fluorescent capabilities fitted with a Zeiss MRC5 digital camera and digital image acquisition software (Axiovision, Carl Zeiss), a Nikon ECLIPSE 80i upright fluorescent microscope equipped with an X-Cite120 Fluorescence Illumination System and a Photometrics CoolSnap digital camera, a Nikon ECLIPSE TS100 inverted microscope, a Nikon DIAPHOT phase-contrast microscope with appropriate camera, a Zeiss LSM 510 confocal microscope and a Nikon A1 confocal system with 408nm, 488nm and 561nm lasers on a TE-2000E2 motorized inverted microscope.

In addition, the Institute of Molecular Cardiology maintains a Forma -152°C ultralow freezer, a liquid nitrogen cryogenic tank, a Forma -80°C freezer and numerous -20°C freezers for sample storage. We also have access to numerous centrifuges, ultracentrifuges, and darkrooms in the Departments of Biochemistry, Nephrology, and Pharmacology and Toxicology.

We also have a mass spectrometer (Waters Technologies, Micromass Platform LCZ 2000 LC-MS) for the analysis of post-translational modification of proteins and enzymes. This is a single quadrapole electrospray mass spectrometer with a nanospray adapter. The spectrometer is attached to a Waters Alliance HPLC with photodiode array detector. The HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESIMS) provides a means for quick and efficient screening of entire protein digests for covalent modifications. The LC/ESIMS analysis yields single-site or multiple-site protonated peptide ions from picomolar quantities of samples. From the m/z value of these ions, the molecular mass of the corresponding peptide can be determined.

IV. Tissue Culture Facilities

The extensive cell culture resources in the Institute of Molecular Cardiology include 6 cell culture laminar flow hoods and 9 incubators in Baxter I and 6 cell culture laminar flow hoods and 16 incubators in Baxter II. These are located in dedicated common cell culture facilities as well as individual labs. In Baxter I, four core culture rooms are located within the Institute of Molecular Cardiology space. Two main culture rooms each contain two laminar flow hoods, incubators, refrigerators, a centrifuge, and a Nikon inverted microscope. One of the main culture rooms accommodates the Viral Vector Core BL-2 facility for the production of recombinant adenovirus and retrovirus. This Viral Vector Core has two six foot Forma Class IIA/B3 biological safety laminar flow hoods, two Forma double upright CO2 water-jacketed incubators, two Nikon inverted microscopes, an Eclipse TE200 with Spot digital camera and a Diaphot 200, both equipped for fluorescent microscopy. The other main culture room contains one 6 foot and one 4 foot Sterilgard Class 2 type A/B3 laminar flow hood, a Nikon inverted microscope, one double upright CO2 water-jacketed incubator, and a single Forma CO2 water-jacketed incubator. Each of the other two culture rooms is adjacent to one of the main laboratory areas and contains one 6-foot hood and CO2 water-jacketed incubator. One of these rooms is also equipped for primary culture of cardiac myocytes and for hypoxia experiments using cultured cells in a Plas Labs Controlled Atmosphere chamber. Similar facilities are available within the Institute of Molecular Cardiology space in the Baxter II Building.

V. Viral Vector Core

The Viral Vector Core, a fully equipped facility within the research space of the Institute of Molecular Cardiology, is located in the Baxter I Building. The Core occupies a total of 1,190 sq. ft. of dedicated space which is organized into three areas: a molecular biology laboratory for vector construction and recombinant virus analysis (732 sq. ft.), a tissue culture room for non-viral work (201 sq. ft.), and a tissue culture room dedicated to viral work (257 sq. ft.).

The lab is fully equipped with state-of-the-art instrumentation for the construction, propagation, and purification of viral vectors. It has two Biosafety Level 2 cabinets, four dual-chamber, water-jacketed tissue culture incubators, two tabletop centrifuges, three refrigerators, four freezers (two -80°C and two -20°C), water baths, an incubator shaker, a Max4000 orbital incubating shaker, an accessible PTC-100 Programmable Thermal Controller (PCR), a Bechman Coulter DU730 spectrophotometer, a Leica CM1900 Cryostat, and three Pentium-class computers for data analysis and lab management. The Core has access to a Sorvall RC26 Plus centrifuge, two Beckman supercentrifuges, and a Beckman LE-80 ultracentrifuge. The facility stores and maintains all of the reagents and general supplies for tissue culture and for viral vector propagation.

VI. Flow Cytometry and Cell Sorting Core

The Institute of Molecular Cardiology owns and maintains a fluorescence-activated cell sorter (MoFlo, Dako Inc., Cupertino, CA) housed within the MDR Building, which is capable of ultrahigh-speed (>50,000 events/second) cell sorting. The MoFlo is housed in a room adjacent to the confocal microscopy laboratory. This MoFlo is equipped with 3 laser sources: 488 nm, 635 nm, and a multiline (351/405/514/568 nm) Krypton laser [Innova 90C-K, Coherent]) for use at multiple excitation wavelengths; 9 PMTs and set of optics for each path; an electronics tower equipped with several logic-boards, dual dot-plot oscilloscope, eight 14-bit analog channels; one Tektronix oscilloscope; 4-way sort kit; Cyclone automated cell cloning accessory; Sort-master automated sample station; a control computer with Windows XP operating system with two 18” high-resolution flat-screen monitors; and the Summit (version 4.3) software. The specifications include: analysis rate of 100,000 eps; sort rate of 70,000 eps; sensitivity < 150 MESF FITC, <100 MESF PE; purity > 99% at all speed; plate deposition 6-1536 wells plus custom configurations; excitation lines up to 3; data resolution up to 5 decades; particle resolution < 0.2 ?m to 25 ?m; and available signal log, height, area, width, and log area for each parameter. The system provides excellent data handling capabilities, high viability and yield of sorted cells, >1 billion event listmode files, auto-compensation, and standard and custom plate sort capabilities. Individual stream deflection can be controlled with simple coarse and fine alignment, droplet control, and stream configuration/control options. The system also allows determination of regions with full parameter resolution, and customized, accurate control of single cell deposition. Other features include CyCLONE calibration and real-time display of sort statistics. Three staff members of the Institute of Molecular Cardiology have received formal training on the operation of this instrument and will process samples for potential users.

Together with the Diabetes and Obesity Center we also operate a Becton Dickinson LSR II located on the second floor of the Baxter II Building. This instrument has 405 nm, 488 nm, and 633 nm lasers and appropriate mirrors, filters and photomultiplier tubes for up to eight color detection. This LSR II has expandable laser and detector capacity, should the need arise for additional color analysis. The system also has a control computer with Windows XP operating system, two 18” high-resolution flat-screen monitors and a Ricoh CL3500N laser color printer. The LSR II is equipped with FacsDiva software but the Institute of Molecular Cardiology also maintains a license for the commonly used FloJo analytical package. LSR II usage will be on an individual basis once appropriate training has been completed with Core personnel.

To meet increasing demand, the Institute of Molecular Cardiology has also recently obtained a Becton Dickinson FASCalibur and an Acuri C6 flow cytometer. Both are available to accommodate more users and, in the case of the FACSCalibur, to provide the potential for clinical purposes. The FASCalibur has 488 nm and 63 nm lasers and 5 detectors, while the Acuri instrument has an identical set of lasers and the capability to detect 6 parameters. Advantages of the latter instrument are its ease of use and portability. Analysis of simple staining protocols can be done on this instrument, allowing the LSR-II to be reserved for more complex, multi-color experiments. All of the cytometers are run from a dedicated computer and their individual software, though analysis can be performed after data export with third party software.

VII. Histology and Pathology Core

The Institute of Molecular Cardiology has a Pathology Core Laboratory located in the Baxter I Building. This facility is capable of processing fixed and frozen samples for sectioning with subsequent staining and histologic and pathological analysis. The Core facility has a pathology laboratory for tissue processing which is equipped with a TBS automated tissue processor for paraffin embedding, a Leica embedding station, a Tissue Tek automated staining station, a motorized Olympus microtome and a TBS cryostat,

The Core also has two microscope rooms for imaging (373 sq ft), and a molecular biology laboratory for biological analysis of CPCs and tissues. The microscope rooms are fully equipped with state-of-the-art instrumentation for pathological analysis of cells and tissues. The Core has 2 cryogenic storage systems, one Nikon DIAPHOT phase-contrast microscope, one Nikon ECLIPSE TE2000-U fluorescent inverted microscope with Zeiss digital camera, one Nikon ECLIPSE 80i fluorescent upright microscope with Photometrics CoolSNAP ES digital camera, and one confocal microscope system (ZEISS LSM 510). Equipment in the molecular biology lab includes two tabletop centrifuges, four refrigerators, three -20°C freezers, two -80°C freezers, water baths, an incubating shaker, one StepOne Plus for real-time PCR, and six computers for image data analysis and laboratory management. The Core has access to a Sorvall RC26 Plus centrifuge, two Beckman supercentrifuges, and a Beckman LE-80 ultracentrifuge. Additional equipment at other sites available for tissue preparation include one Sakura Tissue Processor, one Tissue Embedding System (TES 99), two Leica microtones, and one Leica cryostat (CM1900).

1. Confocal Microscope

Within the Pathology Core, the Institute of Molecular Cardiology owns and maintains a Laser Scanning Module (LSM 510, Carl Zeiss, Inc., Thornwood, NY) housed within the MDR Building. This confocal Laser Scanning module is equipped with one inverted microscope (Axiovert 200); four different laser sources (405 Diode/Argon/HeNe at 543 nm/HeNe at 633 nm) for use at different wavelengths; a set of filters, filter changer; a control computer with Windows 2000 operating system with two 21” CRT monitors; necessary software (LSM 510 and Image Examiner) with multiple possible configurations for image processing and optional software including physiology evaluation software and 3D software. LSM confocal technique projects light (laser) through a high-NA objective onto a certain plane of interest in the object and suppresses light coming from the out-of-focus areas of the specimen. Object features in the order of 0.2?m can be resolved and height difference of less than 0.1?m made visible. The spectral range extends from the visible to the near-ultraviolet. The visible laser module provides wavelengths of 633, 543, 488, and 458 nm while a diode module provides 405 nm wavelengths. Acquired image stacks from different planes can be presented three dimensionally. The acousto-optical tunable filters (AOTF) adjust the necessary brightness for all desired laser lines within microseconds. Four simultaneous image acquisition channels, usable for reflection or fluorescence, and an additional transmitted light channel are ideal for the investigation of multiple fluorescence specimens. An additional digital camera (MRC, Zeiss) is mounted on the Axiovert microscope and images are acquired via Axiovision software available in the same computer.

The Group Picture of the Institute of Molecular Cardiology

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